Mirror nucleic acid replication system

ABSTRACT

Provided is a method for replicating a mirror nucleic acid, comprising: reacting a mirror nucleic acid template, a mirror nucleic acid primer and mirror dNTPs/rNTPs in the presence of a mirror nucleic acid polymerase, so as to obtain the mirror nucleic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International PatentApplication No. PCT/CN2017/073573, filed Feb. 15, 2017, andPCT/CN2016/079006, filed Apr. 11, 2016, entitled “MIRROR NUCLEIC ACIDREPLICATION SYSTEM”, and the contents of which are herein incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering, andmore particularly to the replication and transcription of a mirror-imagenucleic acid.

BACKGROUND OF THE INVENTION

Chirality is a basic property of some molecules in three-dimensionalspace, that is, an object cannot coincide with its image in the mirror.Like the left and right hands of a person, the two are in a relationshipof an object and its mirror-image and cannot overlap regardless of howthey are flipped in three-dimensional space. This is the chirality of anobject in space. A mirror-image of a molecule is called its enantiomer.They have the same physical and chemical properties, and have the samemelting point, molecular weight, solubility, density, and NMR spectrum.The mutually different nature of the enantiomers is their opticalproperty—rotating the direction of a plane polzaried light. Chiralitywidely exists in nature. Biomacromolecules in living organisms such asproteins, polysaccharides, DNA and RNA are chiral. For all organisms onthe earth, whether large plants, mammals or microorganisms that areinvisible to the naked eye, among the 20 amino acids that constitute theprotein, except for the fact that glycine has no chirality, the other 19amino acids are all in L-form; while for DNA and RNA carrying geneticinformation, their riboses are all in D-form.

The amino acid has a chiral carbon atom in the immediate vicinity of thecarboxyl group. As a chiral center, it makes the amino acids other thanglycine have both L and D chirality. L refers to levorotatory orleft-handed, and D refers to dextrorotatory or right-handed. For nucleicacids, D-form is the chirality found in nature, and the chiral center ofa nucleic acid molecule is located on its backbone.

The biological activities of two compounds with different chirality in aliving organism may be completely different. Enzymes and cell surfacereceptors in biological individuals are mostly chiral, and the twoenantiomers are often absorbed, activated and degraded in different waysin the organism. For a common chiral drug, either the two enantiomersmay have equivalent pharmacological activities, or one may be active,but the other may be inactive, even toxic. At present, it is believedthat the molecules of proteins and nucleic acids present in livingorganisms have the characteristics of chiral unitarity. If a nativeprotein sequence is incorporated with a mirror-image amino acid, its ownsecondary structure will be destroyed (Krause et al., 2000), and itsprotein function will be severely affected.

In early environment of the earth, there should be the presence ofbiomolecules such as amino acids and nucleic acids before the mostprimitive cells appeared. Although the theory of RNA as the origin ofliving matter is well-established, there is currently no evidence ofwhich of the amino acid and nucleic acid first appeared on the earth.The study of the meteorite of Murchison, which fell in Australia in1969, found that it contained a variety of amino acids, includingglycine, alanine and glutamic acid. The two kinds of chiral amino acidswere not 1:1, and the L-amino acid was more than the D-amino acid (Engeland Macko, 2001). Experiments by Breslow and Levine showed that evenwith small chiral difference, it was possible to retain more than 90% ofa chiral amino acid in solution after two times of evaporation andcrystallization of the solution (Breslow and Levine, 2006), and thisprocess could occur in the early environment of the earth. In addition,there are other hypotheses that the chiral unitarity in evolution wasderived from the optically active crystal generated by calcite thatselectively adsorbed an optically active amino acid, increasing theproportion of the other in solution. In addition to the above twoexplanations, the current RNA origin of life hypothesis believes thatRNA was formed by organic molecules in the early environment of theearth, and RNA had evolved into a biologically functional RNA, an RNAribozyme, which had activity such as self-replication and self-cleavage;in further evolution, proteases or peptides composed of amino acidsbegan to participate in catalyzing the replication, unwinding, and thelike of RNA, and there might be a process of undergoing chiral selectionduring this stage, which led to the chiral unitarity in the complexliving organisms that finally evolved.

SUMMARY OF THE INVENTION

In this study, we constructed a genetic information transcription andreplication system based on D-ASFV pol X mirror-image polymerase bychemical synthesis, and realized two steps in the mirror-image centerrule: replication of L-DNA and transcription into L-RNA. We confirmedthat the replication and transcription of mirror-image DNA followed thebase complementary pairing rules and have good chiral specificity. Wefound that when the natural and mirror-image DNA replication systemswere placed in the same solution system, the two could work separatelywithout serious mutual interference. The mirror-image polymerase systemamplifies the L-DNAzyme deoxyribozyme sequence to achieve self-splicingactivity corresponding to the native deoxyribozyme. The realization ofmirror-image genetic information replication and transcriptionillustrates the potential for existence and biological activity ofmirror-image life molecules, and also lays the foundation for the futureconstruction of mirror-image cells in the laboratory environment. Afterfurther optimization of the system, the mirror-image replication systemwill also be used for efficient screening of a mirror-image nucleic aciddrug by a biological method.

The present invention provides a method for replicating a mirror-imagenucleic acid comprising: carrying out a reaction in the presence of amirror-image nucleic acid polymerase, a mirror-image nucleic acidtemplate, a mirror-image nucleic acid primer, and mirror-imagedNTPs/rNTPs to obtain the mirror-image nucleic acid.

The present invention provides a method for performing mirror-image PCR,comprising: carrying out a reaction in the presence of a mirror-imagenucleic acid polymerase, a mirror-image nucleic acid template, amirror-image nucleic acid primer, and mirror-image dNTPs/rNTPs to obtaina mirror-image nucleic acid.

The present invention provides a method for screening a mirror-imagenucleic acid molecule comprising: contacting a library of randommirror-image nucleic acid sequences with a target molecule under acondition that allows binding of the two; obtaining a mirror-imagenucleic acid molecule that binds to the target molecule; and amplifyingthe mirror-image nucleic acid molecule that binds to the target moleculeby mirror-image PCR.

The present invention also provides D-ASFV pol X, the sequence of whichis set forth in SEQ ID NO: 17, wherein except for glycine which is notchiral, all other amino acids are D-form amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemical synthesis route of D-ASFV Pol X. TheASFV pol X of 174 amino acids was synthesized and ligated by threesegments: peptide segment 1: Met1-Lys85, peptide segment 2: Cys86-Leu105and peptide segment 3: Ala106-Leu174. Cys86 was first protected withAcm, Cys86-Leu105 was synthesized, and then peptide segment 3:Cys106-Glu107-Leu174 was synthesized. After activation and ligation, theligation product of peptide segment 2 and peptide segment 3 wasobtained, and then Cys106 was desulfurized to form Ala106. Then peptidesegment 1 was synthesized, the Acm protecting group of peptide segment 2was catalytically removed, and activation and ligation were performed toobtain full-length ASFV pol X polymerase.

FIG. 2 illustrates the detection of the full-length product of D-ASFVpol X synthesis. a. HPLC spectrum of D-ASFV pol X after folding. HPLCanalysis used a absorption wavelength of 214 nm, and a Vydac C18(4.6×250 mm) LC column. b. ESI-MS spectrum, by analyzing and calculatingthe ion peak map, the size of the main synthetic product was observed tobe 20317.0 Da, while the theoretical value of ASFV pol X was 20316.0 Da.

FIG. 3 illustrates L- and D-ASFV pol X detection. a. SDS-PAGE detection,E. coli-expressed, chemically synthesized L-ASFV pol X polymerase andchemically synthesized D-ASFV pol X polymerase were separated on 15%SDS-PAGE gel and detected by silver staining. M, protein molecularweight marker. b. CD detection, L-form and D-form chemically synthesizedASFV pol X were tested on an Applied Photophysics Pistar-180 CDspectrometer, and the absorption curve was the average of threeindependent tests subtracting the background.

FIG. 4 illustrates a mirror-image DNA assay. a. HPLC detection (providedby Chemgenes), the purification and analysis of four L-dNTPs werecarried out by HPLC, the results of L-dATP and L-dGTP showed almost noimpurity peaks, while L-dTTP and L-dCTP had obvious impurity peaks. b.Primer12 CD detection, for the commercially available HPLC purifiedL-form and PAGE purified D-form primer12, the absorption curves had asymmetrical relationship (the actual concentration of L-primer12 wasless than that of D-primer). c. Template18 CD detection, for thecommercially available L-form and D-form template18, the absorptioncurves in the CD detection had a symmetric relationship (the loadingconcentration of the sample of L-template18 was actually less than thatof D-primer).

FIG. 5 illustrates the DNA extension catalyzed by a mirror-imagepolymerase. a. Schematic diagram of natural and mirror-image DNAreplication systems, the natural DNA replication system comprisedL-polymerase, D-DNA and D-dNTPs; mirror-image DNA replication systemcomprised D-polymerase, L-DNA and L-dNTPs. b. DNA extension catalyzed bynatural and mirror-image polymerase (12 nt primer, 18 nt template), thebuffer conditions were 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT,and 50 mM KCl, and to a 10 μl natural or mirror-image reaction system, acorresponding chiral 0.7 μg of ASFV pol X, 2.5 μM primer 12, 2.5 μMtemplate 18 and four kinds of 0.4 mM dNTPs were added. The reactionsystem was placed at 37° C. for 4 hours. “*” indicates a 5′FAM mark. c.ESI-MS detection of L-DNA full-length product, the expected molecularweight of the full-length product was 5516.9 (theoretical value of5517.6) and the molecular weight of the template was 5481.9 (theoreticalvalue of 5481.6) by analytical calculation. d. DNA extension catalyzedby natural and mirror-image polymerase (15 nt primer, 18 nt template),the reaction system was carried out in 50 mM Tris-HCl pH 7.5, 20 mMMgCl₂, 1 mM DTT, and 50 mM KCl. And 2.5 μM 15-nt L-primer15 (without FAMmodification), 2.5 μM 21-nt L-template21, 0.2 mM L-dNTPs (theconcentration for each kind), and 1.4 μg of D-ASFV pol X were added.After 12 hours of reaction at 37° C., separation was performed by 20%PAGE gel and detection was performed by Sybr Gold staining.

FIG. 6 illustrates a schematic diagram of multi-cycle amplification ofmirror-image DNA. In the first cycle, the reverse 11 primer without FAMis amplified to obtain a double-stranded template. In the second cycle,the FAM-labeled primer11 can only complement to the template amplifiedby the first cycle (orange-red) to obtain the full-length product. Inthe third cycle, the fluorescent full-length product is produced in anamount three times that of the second cycle.

FIG. 7 illustrates multi-cycle amplification of a mirror-image DNAreplication system. D-ASFV pol X amplifies L-DNA in multiple cycles,cycle 0 was the control group and was sampled before cycle 1 wasperformed. Cycle 1 amplification yielded the full-length product ofreverse11, which could be used as a template by subsequent cycles.Samples were separated on 8 M urea denatured 20% PAGE gel and scannedfor fluorescence with Typhoon Trio+. All products detected wereFAM-labeled DNA.

FIG. 8 illustrates the base complementary pairing specificity of themirror-image DNA extension. One of the corresponding chiral 0.2 mM dATP,dTTP, dCTP, and dGTP was added to the natural or mirror-image system.The next base of the template was A, T, C, G (white box mark on blackbackground), buffer conditions were 50 mM Tris-HCl pH 7.5, 20 mM MgCl₂,1 mM DTT, and 50 mM KCl. And a corresponding chiral 0.7 μg of ASFV polX, 2.5 μM primer, 2.5 μM template18 were added. The reaction was carriedout at 37° C. for 30 minutes, electrophoresis was performed on a 20%PAGE gel and the fluorescent signal was scanned with a Typhoon Trio+scanner. “*” indicates a 5′FAM mark. “-” indicates a control group towhich D or L-dNTPs were not added.

FIG. 9 illustrates the chiral specificity of mirror-image DNA extension.In buffer conditions of 50 mM Tris-HCl pH 7.5, 20 mM MgCl₂, 1 mM DTT,and 50 mM KCl, 0.7 μg of ASFV pol X, 2.5 μM primer, and 2.5 μM template18 were added. The chirality of the protein, primer-template, and dNTPsare listed below the figure, and there is a total of 8 combinations. Thereaction was carried out at 37° C. for 12 hours, electrophoresis wasperformed on a 20% PAGE gel and the fluorescent signal was scanned witha Typhoon Trio+ scanner. “*” indicates a 5′FAM mark.

FIG. 10 illustrates the reactions of the natural and mirror-imagesystems in the same solution. In buffer conditions of 50 mM Tris-HCl pH7.5, 20 mM MgCl₂, 1 mM DTT, and 50 mM KCl, 0.7 μg of each of the twochiral ASFV pol X, 2.5 μM natural 5′-end Cy5 labeled primer20, 2.5 μMnatural template26, 2.5 μM mirror-image 5′-end FAM-labeled primer12, 2.5μM mirror-image template18, and four kinds of dNTPs for each kind ofcharility, each with a final concentration of 0.2 mM, were added. Thereaction system was reacted at 37° C. for 4 hours, and the reactionproducts were separated on 8 M urea denatured 20% PAGE gel and scannedwith Typhoon Trio+ in Cy5 and FAM fluorescence modes, and the pictureswere combined together.

FIG. 11 illustrates the enzyme synthesis and activity assay of nativeand mirror-image DNAzymes. a. Zn2+-dependent DNAzyme secondarystructure. A 12 nt primer sequence was added to the 5′ end of the 44 ntDNAzyme to a full-length of 56 nt. The secondary structure was generatedby the mfold server (Zuker, 2003). b. Enzymatic synthesis of DNAzyme.Both native and mirror-image DNAzymes were reacted in a buffer of 50 mMTris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT, and 50 mM KCl, and 66 ntDNAzyme template and 12 nt primers were added and reacted at 37° C. for36 hours to obtain a full-length product. M, the marker was a chemicallysynthesized 56 nt sequence as a molecular weight standard. c. Theextended full-length DNAzyme was excised from the PAGE gel, spread in abuffer overnight, and precipitated by a Tiandz PAGE gel recovery kit.The precipitated DNA was dissolved in a buffer of 1:50 mM HEPES, pH 7.0and 100 mM NaCl, and heated at 90° C. for 2 min, and then cooled on icefor 5 min. An equal volume of a buffer of 2:50 mM HEPES, pH 7.0, 100 mMNaCl, and 4 mM ZnCl₂ or 40 mM MgCl₂ was then added to initiate thereaction (final concentrations of Zn²⁺ and Mg²⁺ were 2 mM and 20 mM).The reaction was carried out at 37° C. for 36 hours. Finally, thereaction was terminated with EDTA. The samples were separated anddeveloped on a 12% PAGE gel.

FIG. 12 illustrates DNA template-dependent RNA transcription of thenative and mirror-image systems. a. Native and mirror-image ASFV pol Xcatalyzed RNA transcription. Buffer conditions were 50 mM Tris-HCl, pH7.5, 20 mM MgCl₂, 1 mM DTT, and 50 mM KCl. To a 10 μl reaction system,0.7 μg of ASFV pol X, 2.5 μM primer, 2.5 μM template and four kinds of0.4 mM rNTPs were added. 2 units of RNase inhibitor was added to thenatural system. The reaction was terminated after reacting at 37° C. for60 hours. b. The full-length product was obtained after 36 hours ofreaction at 37° C. in the natural system with rNTP added. ASFV pol X andRNase inhibitors were inactivated by heating to 75° C. for 10 min. 1μg/μl, 0.1 μg/μl, and 0.01 μg/μl of RNase A were added to each of thethree experiments, followed by incubation at 23° C. for 10 min. Thedegradation reaction was terminated by addition of 20 units of RNaseinhibitor and a loading buffer was added. Reaction products wereseparated on 8 M urea denatured 20% PAGE gel and imaged using theTyphoon Trio+ system. Sample 1: Control group, extended for 0 hours;Sample 2: The full-length product of D-primer12 extended over 36 hours;Sample 3: The full-length product heated at 75° C. for 10 minutes toinactivate RNase inhibitor and ASFV pol X; Sample 4-6: The full-lengthextension products were heated at 75° C. for 10 minutes, 0.01 μg/μl, 0.1μg/μl, or 1 μg/μl of RNase A was added, respectively, and the mixtureswere allowed to stand at 23° C. for 10 minutes, and the reaction wereterminated by adding 20 units of RNase inhibitor. Electrophoresis wasperformed on a 20% PAGE gel and the fluorescent signal was scanned witha Typhoon Trio+ scanner. “*” indicates a 5′FAM mark.

FIG. 13 illustrates the base complementary pairing specificity of nativeand mirror-image system RNA transcription. In the natural ormirror-image system, one of the corresponding chiral 0.2 mM rATP, rUTP,rCTP, and rGTP was respectively added, and the next base of the templatewas A, T, C, and G (white box mark on black). Buffer conditions were 50mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT, and 50 mM KCl. 0.7 μg ofcorresponding chiral ASFV pol X, 2.5 μM primer, 2.5 μM template18 wereadded. The reaction was carried out at 37° C. for 12 hours,electrophoresis was performed on a 20% PAGE gel and the fluorescentsignal was scanned with a Typhoon Trio+ scanner. “*” indicates a 5′FAMmark. “-” indicates a control group to which D or L-dNTPs were notadded.

FIG. 14 illustrates the total chemical synthesis design of mutant Dpo4polymerase. (a) The amino acid sequence of wild-type Dpo4 polymerase(SEQ ID NO: 45); (b) The amino acid sequence of His6-tagged mutant Dpo4polymerase (Dpo4-5m) corresponding to SEQ ID Nos: 46 and 47, wherein thehighlighted amino acid sites were the four mutations introduced in orderto increase the number of peptide ligation sites (S86A, N123A, 5207A andS313A), and a mutation site (C31S) introduced to prevent intramoleculardimerization caused by disulfide bond formation during folding,respectively. Furthermore, all methionines in the peptide chain werereplaced with norleucine (Met1, Met76, Met89, Met157, Met216 and Met251)to prevent oxidation during solid phase peptide synthesis and peptideligation. The colors of the nine peptide sgements in this figurecorrespond to the colors used in the peptide segments (Dpo4-1 to Dpo4-9)in FIG. 2.

FIG. 15 illustrates the synthetic route of Dpo4-5m. The hydrazide wasused instead of the thioester to mediate the assembly of the ninepeptide segments in the direction from the C to the N-terminal tofinally achieve the total chemical synthesis of Dpo4-5m.

FIG. 16 illustrates the biochemical properties of Dpo4-5m polymerase.(a) The molecular weight and purity of the chemically synthesizedDpo4-5m polymerase and purified recombinant Dpo4-5m polymerase expressedfrom E. coli strain BL21 (DE3) were analyzed using 12% SDS-PAGE, andstained with Coomassie blue. A small amount of unligated peptide wasobserved in the synthesized Dpo4-5m. M was a standard molecular weightprotein marker. (b) A 200 bp DNA sequence was PCR amplified usingrecombinant wild-type Dpo4 (labeled as “WT Dpo4” in the figure),recombinant mutant Dpo4-5m (“Recombinant”) and synthetic mutant Dpo4-5m(“Synthetic”) polymerase. The PCR reaction system contained 50 mM HEPES(pH 7.5), 5 mM MgCl₂, 50 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% glycerol,3% DMSO, 0.1 mg/ml BSA, 200 μM ultrapure dNTPs, 0.5 μM bidirectionalprimer, 2 nM linear double stranded DNA template and approximately 300nM Dpo4-5m enzyme. After 35 cycles of reaction, the PCR product wasanalyzed by 2% agarose gel electrophoresis and stained with GoldView(Solarbio). NC was a negative control experiment in which only templateand primer were present in the reaction system, and no polymerase. (c)The 200 bp DNA products were obtained from different cycles ofamplication using synthetic Dpo4-5m. The PCR products were analyzed by2% agarose gel electrophoresis and stained with GoldView (Solarbio), andthe numbers above the lane represent the numbers of cycles ofamplification. M was a standard molecular weight DNA marker.

FIG. 17 illustrates the PCR amplification of DNA sequences of differentlengths using chemically synthesized Dpo4-5m polymerase. (a) Thesynthesized Dpo4-5m polymerase was used to PCR amplify sequences withlengths from 110 bp to 1 kb, with 35 cycles per reaction. The extensiontime for each cycle of amplification of the 110-300 bp sequence was setto 2 minutes, the sequence of 400-600 bp was extended for 5 minutes, andthe sequence of 700-1000 bp was extended for 10 minutes. The expectedlength of the amplicon is indicated above the lane and a primer dimerband can be seen below the main product band. The W/o template indicatesa negative control without a template, and the reaction only had primer,enzyme and other PCR components. NC (110 bp)/NC (1 kb) indicates anegative control without enzyme addition, and the reaction only hadprimer, template (110 bp/1 kb) and other PCR components. (b) Thesynthesized Dpo4-5m polymerase was used to PCR amplify a 1.1 kb dpo4gene, with 35 cycles per reaction, and the extension time in each cyclewas set to 10 minutes. (c) The synthesized Dpo4-5m polymerase was usedto PCR amplify the 120 kb 5S rRNA gene rrfB in E. coli, with 35 cyclesper reaction, and the extension time in each cycle was set to 2 minutes.(d) The synthesized Dpo4-5m polymerase was used to PCR amplify the 1.5kb 16S rRNA gene rrfC in E. coli, with 35 cycles per reaction, and theextension time in each cycle was set to 15 minutes. A primer dimer maybe observed under the main product band. All PCR products were analyzedby 2% agarose gel electrophoresis and stained with GoldView. Theexpected amplicon length is indicated above the lane, NC represents thenegative control without enzyme addition, and only the primer andtemplate and other PCR components were present in the reaction, and Mwas a standard molecular weight DNA marker.

FIG. 18 illustrates the assembly PCR using the synthetic Dpo4-5mpolymerase. (a, b) The PCR was carried out by two long primers(tC19Z-F115 and tC19Z-R113) sharing a 30 bp overlap region. Theamplified target product was 198 bp tC19Z gene, and the reaction wascarried out for 20 cycles. The PCR product was analyzed by 3% highresolution agarose gel electrophoresis and stained with GoldView(Solarbio). Exo I indicates treatment with exonuclease I, which digestsonly single-stranded DNA without digesting double-stranded DNA. (c, d)Three-step PCR reactions were performed with six short primers rangingin lengths from 47 nt to 59 nt, and the 198 bp long tC19Z gene wasassembled. The PCR product was analyzed by 3% high resolution agarosegel electrophoresis and stained with GoldView (Solarbio). The numbers ofcycles of the first, second, and third step of the PCR reaction were 5,10, and 20, respectively. The sequence lengths expected to be amplifiedin each of the three steps of the PCR reactions were 88 bp, 162 bp, and198 bp, respectively (labeled above the lane). A primer dimer may beobserved under the main product band and M was a standard molecularweight DNA marker.

FIG. 19 illustrates the preparation of Dpo4-10. (A) 54 mg of Dpo4-1 wasdissolved in 2 ml of an acidified ligation buffer (6 M Gn.HCl and 0.1 MNaH₂PO₄ in water, pH 3.0). The mixture was placed in an ice salt bathand cooled, then 200 μl of an acidified ligation buffer containing 0.5 MNaNO₂ was added, and after stirring for 30 minutes in an ice salt bath,2 ml of 0.2 M MPAA (dissolved in 6 M Gn.HCl and 0.1 M Na₂HPO₄, pH 6.0)was added. Then 49 mg of Dpo4-2 was added and the pH of the solution wasadjusted to 6.5 with sodium hydroxide solution at room temperature.After 15 hours of reaction, reduction with tris(2-carboxyethyl)phosphinefollowed by HPLC purification gave 52 mg of Dpo4-10 with a yield of 51%;(B) Analytical high performance liquid chromatogram of Dpo4-10 (λ=214nm), column: Welch C4; gradient: mobile phase ratio CH₃CN:H₂O from 20%to 70% (both with 0.1% TFA), elution for more than 30 minutes; (C)Electrospray ionization mass spectrum of Dpo4-10.

FIG. 20 illustrates the preparation of Dpo4-11. (A) 43 mg of Dpo4-10 wasdissolved in 2 ml of an acidified ligation buffer (6 M Gn.HCl and 0.1 MNaH₂PO₄ in water, pH 3.0). The mixture was placed in an ice salt bathand cooled, then 160 μl of an acidified ligation buffer containing 0.5 MNaNO₂ was added, and the system was stirred in an ice salt bath for 25minutes, and then 24 mg of sodium sulfonate (MESNa) was added. The pH ofthe solution was adjusted to 5.1 with sodium hydroxide solution at roomtemperature. After 1 hour of reaction, the reaction product was purifiedby HPLC to finally give 25 mg of Dpo 4-11 with a yield of 60%. (B)Analytical high performance liquid chromatogram of Dpo4-11 (λ=214 nm),column: Welch C4; gradient: mobile phase ratio CH₃CN:H₂O from 20% to100% (both with 0.1% TFA), elution for more than 30 minutes; (C)Electrospray ionization mass spectrum of Dpo4-11.

FIG. 21 illustrates the preparation of Dpo4-12. (A) 45 mg of Dpo4-3 wasdissolved in 4 ml of an acidified ligation buffer (6 M Gn.HCl and 0.1 MNaH₂PO₄ in water, pH 3.0). The mixture was placed in an ice salt bathand cooled, then 200 μl of an acidified ligation buffer containing 0.5 MNaNO₂ was added and stirred in an ice salt bath for 25 minutes, then 1ml of an acidified ligation buffer containing 0.4 M sodium sulfonate(MESNa) was added. The pH of the solution was adjusted to 5.1 withsodium hydroxide solution at room temperature. After 1 hour of reaction,the product was purified by HPLC to finally give 19 mg of Dpo4-12 with ayield of 41%; (B) Analytical high performance liquid chromatogram ofDpo4-12 (λ=214 nm), column: Welch C4; gradient: mobile phase ratioCH₃CN:H₂O from 20% to 70% (both with 0.1% TFA), elution for more than 30minutes; (C) Electrospray ionization mass spectrum of Dpo4-12.

FIG. 22 illustrates the preparation of Dpo4-13. (A) 70 mg of Dpo4-5 wasdissolved in 2.4 ml of an acidified ligation buffer (6 M Gn.HCl and 0.1M NaH₂PO₄ in water, pH 2.9). The mixture was placed in an ice salt bathand cooled, then 240 μl of an acidified ligation buffer containing 0.5 MNaNO₂ was added and stirred in an ice salt bath for about 30 minutes,then 2.4 ml of 0.2 M MPAA (dissolved in 6 M Gn.HCl and 0.1 M Na₂HPO₄, pH4.9) was added. The pH of the solution was adjusted to 5.4 with sodiumhydroxide solution at room temperature, and after addition of 36 mg ofDpo4-6, the pH of the solution was again adjusted to 6.6. After 11 hoursof reaction, the product was analyzed and the pH was adjusted to 9.0 tocompletely remove the Tfa group. The product was analyzed again afterabout 1 hour, and then 72 mg of MeONH₂.HCl was added to achieveconversion of Thz to Cys, and the pH of the reaction system was adjustedto 4.0 by the addition of TCEP.HCl. After about 3 hours, the product wasanalyzed and purified by HPLC to give 56 mg of Dpo 4-13 with a yield of57%. (B) Analytical high performance liquid chromatogram of Dpo4-13(λ=214 nm), column: Welch C4; gradient: mobile phase ratio CH₃CN:H₂Ofrom 20% to 70% (both with 0.1% TFA), elution for more than 30 minutes;(C) Electrospray ionization mass spectrum of Dpo4-13.

FIG. 23 illustrates the preparation of Dpo4-14. (A) The ligationreaction of Dpo4-8 (53 mg) and Dpo4-9 (48 mg) was similar to that ofDpo4-5 and Dpo4-6, see FIG. 22. Finally, 46 mg of Dpo4-13 was obtainedwith a yield of 46%; (B) Analytical high performance liquid chromatogramof Dpo4-14 (λ=214 nm), column: Welch C4; gradient: mobile phase ratioCH₃CN:H₂O from 20% to 100% (both with 0.1% TFA), elution for more than30 minutes; (C) Electrospray ionization mass spectrum of Dpo4-14.

FIG. 24 illustrates the preparation of Dpo4-15. (A) 36 mg of Dpo4-4 wasdissolved in 2 ml of an acidified ligation buffer (6 M Gn.HCl and 0.1 MNaH₂PO₄ in water, pH 3.0). The mixture was placed in an ice salt bathand cooled, then 400 μl of an acidified ligation buffer containing 0.5 MNaNO₂ was added, and stirred in an ice salt bath for 30 minutes, then 2ml of 0.2 M MPAA (dissolved in 6 M Gn.HCl and 0.1 M Na₂HPO₄, pH 6.0) wasadded. After addition of 81 mg of Dpo4-13, the pH of the solution wasadjusted to 6.5 with sodium hydroxide solution at room temperature.After 15 hours of reaction, the product was reduced withtris(2-carboxyethyl)phosphine and purified by HPLC to give 54 mg ofDpo4-15 with a yield of 46%; (B) Electrospray ionization mass spectrumof Dpo4-15.

FIG. 25 illustrates the preparation of Dpo4-16. (A) 82 mg of Dpo4-15 wasdissolved in 7 ml of an acidified ligation buffer (6 M Gn.HCl and 0.1 MNaH₂PO₄ in water, pH 3.1). The mixture was placed in an ice salt bathand cooled, then 140 μl of an acidified ligation buffer containing 0.5 MNaNO₂ was added and stirred in an ice salt bath for 30 minutes, then1.75 ml of an acidified ligation buffer containing 0.8 M sodiumsulfonate (MESNa) was added. The pH of the solution was adjusted to 5.5with sodium hydroxide solution at room temperature. After 2 hours ofreaction, the product was reduced with tris(2-carboxyethyl)phosphine andpurified by HPLC to give 69 mg of Dpo4-16 with a yield of 83%; (B)Analytical high performance liquid chromatogram of Dpo4-16 (λ=214 nm),column: Welch C4; gradient: mobile phase ratio CH₃CN:H₂O from 20% to 70%(both with 0.1% TFA), elution for more than 30 minutes; (C) Electrosprayionization mass spectrum of Dpo4-16.

FIG. 26 illustrates the preparation of Dpo4-17. (A) The ligationreaction of Dpo4-7 (25 mg) and Dpo4-14 (47 mg) was similar to that ofDpo4-5 and Dpo4-6, see FIG. 22. Finally, 41 mg of Dpo4-17 was obtainedwith a yield of 59%; (B) Analytical high performance liquid chromatogramof Dpo4-17 (λ=214 nm), column: Welch C4; gradient: mobile phase ratioCH₃CN:H₂O from 20% to 70% (both with 0.1% TFA), elution for more than 30minutes; (C) Electrospray ionization mass spectrum of Dpo4-17.

FIG. 27 illustrates the preparation of Dpo4-18. (A) 37 mg of Dpo4-16 and41 mg of Dpo4-17 were dissolved in 1.1 ml of an aqueous solution(containing 6 M Gn.HCl, 0.1 M Na₂HPO₄, 40 mM TCEP and 125 mM MPAA, pH6.8), the pH of the reaction mixture was adjusted to 6.6. After 12 hoursof reaction, the product was analyzed by HPLC and then diluted andpurified to finally give 55 mg of Dpo4-18 with a yield of 75%; (B)Analytical high performance liquid chromatogram of Dpo4-18 (λ=214 nm),column: Welch C4; gradient: mobile phase ratio CH₃CN:H₂O from 30% to 80%(both with 0.1% TFA), elution for more than 30 minutes; (C) Electrosprayionization mass spectrum of Dpo4-18.

FIG. 28 illustrates the preparation of Dpo4-19. (A) Removal of the Acmgroup in Dpo4-18 by Pd-assisted deprotection [1], 55 mg of Dpo4-18 wasdissolved in 2 ml of aqueous solution (containing 6 M Gn.HCl, 0.1 MNa₂HPO₄ and 40 mM TCEP, pH 7.1), and then 0.4 ml of an aqueous solution(containing 6 M Gn.HCl and 0.1 M Na₂HPO₄) in which 10.4 mg of PdCl₂ wasdissolved was added to the reaction system. After 13 hours of reaction,4 ml of an aqueous solution of 0.75 M DTT (containing 6 M Gn.HCl and 0.1M Na₂HPO₄) was added, and the reaction mixture was stirred for 1 hour,and the product was purified by HPLC. Finally, 47 mg of Dpo4-19 wasobtained with a yield of 86%; (B) Analytical high performance liquidchromatogram of Dpo4-19 (λ=214 nm), column: Welch C4; gradient: mobilephase ratio CH₃CN:H₂O from 30% to 80% (both with 0.1% TFA), elution formore than 30 minutes; (C) Electrospray ionization mass spectrum ofDpo4-19.

FIG. 29 illustrates the preparation of Dpo4-20. (A) 19 mg of Dpo 4-12and 47 mg of Dpo4-19 were dissolved in 1.8 ml of an aqueous solution(containing 6 M Gn.HCl, 0.1 M Na₂HPO₄, 40 mM TCEP and 100 mM MPAA, pH6.9), and the pH of the reaction mixture was adjusted to 6.6. After 15hours of reaction, the product was analyzed by HPLC, followed bydilution and purification to finally give 47 mg of Dpo4-20 with a yieldof 85%; (B) Analytical high performance liquid chromatogram of Dpo4-20(λ=214 nm), column: Welch C4; gradient: mobile phase ratio CH₃CN:H₂Ofrom 30% to 80% (both with 0.1% TFA), elution for more than 30 minutes;(C) Electrospray ionization mass spectrum of Dpo4-20.

FIG. 30 illustrates the preparation of Dpo4-21. (A) 24 mg of Dpo4-20 wasdissolved in 1.6 ml of acetic acid solution, then 10 mg of silveracetate was added to the solution, and after stirring for 14 hoursovernight, 0.3 ml of 2-mercaptoethanol was added. The reaction systemwas diluted to twice with a ligation solution (6 M Gn.HCl, 0.1 MNa₂HPO₄, pH=7), and the supernatant after centrifugation was purified bysemi-preparative HPLC, and the precipitate was thoroughly washed andpurified. After lyophilization, 20 mg of Dpo4-21 from which the Acmgroup had been removed was obtained with a yield of 83%; (B) Analyticalhigh performance liquid chromatogram of Dpo4-21 (λ=214 nm), column:Welch C4; gradient: mobile phase ratio CH₃CN:H₂O from 20% to 100% (bothwith 0.1% TFA), elution for more than 30 minutes; (C) Electrosprayionization mass spectrum of Dpo4-21.

FIG. 31 illustrates the preparation of Dpo4-22. (A) 13 mg (2 eq.) ofDpo4-11 and 20 mg of Dpo4-21 were dissolved in 0.4 ml of an aqueoussolution (containing 6 M Gn.HCl, 0.1 M Na₂HPO₄, 40 mM TCEP and 100 mMMPAA, pH 6.9), and the pH of the reaction mixture was adjusted to 6.6.After 15 hours of reaction, the product was analyzed by HPLC, followedby dilution and purification to finally give 21 mg of Dpo4-22 with ayield of 78%; (B) Analytical high performance liquid chromatogram ofDpo4-22 (λ=214 nm), column: Welch C4; gradient: mobile phase ratioCH₃CN:H₂O from 20% to 100% (both with 0.1% TFA), elution for more than30 minutes; (C) Electrospray ionization mass spectrum of Dpo4-22.

FIG. 32 illustrates the preparation of Dpo4-23 (Dpo4-5m). (A) 20 mg ofDpo4-22 was dissolved in 6 ml of 200 mM TCEP solution (containing 6 MGn.HCl, 0.2 M Na₂HPO₄, pH 6.9), followed by addition of 0.1 mmol (32 mg)of VA-044 and 0.2 mmol (62 mg) of reduced L-glutathione. The reactionwas stirred at 37° C. overnight. The desulfurized product Dpo4-23 wasanalyzed by HPLC and ESI-MS and purified by semi-preparative HPLC.Finally, 16 mg of lyophilized Dpo4-23 was obtained with a yield of 80%.(B) Analytical high performance liquid chromatogram of Dpo4-23 (λ=214nm), column: Welch C4; gradient: mobile phase ratio CH₃CN:H₂O from 20%to 100% (both with 0.1% TFA), elution for more than 30 minutes; (C)Electrospray ionization mass spectrum of Dpo4-23. (D) Electrosprayionization mass spectrum of Dpo4-23 after further renaturation, heatingand purification on a nickel column with a yield of 15% (approximately 2mg of final product).

FIG. 33 illustrates a PCR-amplified 200-bp sequence using syntheticDpo4-5m PCR, and samples were taken sequentially from each of 0 to 10thcycles.

The PCR product was analyzed by 2% agarose gel electrophoresis andstained with GoldView. The number of cycles sampled is labeled above thelane and M was the DNA marker.

FIG. 34 illustrates the alignment of the Sanger sequencing results ofthe PCR amplified sequences with the original template sequence. The PCRproduct was cloned into the T vector, and positive colonies were pickedfor Sanger sequencing. There were 7 single base deletions and 19 singlebase mutations, indicating that the cumulative mutation rate after 35cycles was about 0.9%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for replicating a mirror-imagenucleic acid comprising: carrying out a reaction in the presence of amirror-image nucleic acid polymerase, a mirror-image nucleic acidtemplate, a mirror-image nucleic acid primer, and mirror-imagedNTPs/rNTPs to obtain the mirror-image nucleic acid.

The term “mirror-image” as used herein, refers to an isomer that is in amirror-image relationship with the natural material in chirality.

The term “mirror-image nucleic acid” as used herein, refers to an L-formnucleic acid that is in a mirror-image relationship with a naturalnucleic acid (ie, a D-form nucleic acid). The mirror-image nucleic acidincludes L-form DNA and L-form RNA. The term “mirror-image DNA” is usedinterchangeably with “L-form DNA” or “L-DNA”.

The term “mirror-image nucleic acid polymerase” or “mirror-imagepolymerase” as used herein refers to a D-form polymerase that is in amirror-image relationship with a native polymerase (ie, an L-formpolymerase). The term “mirror-image polymerase” is used interchangeablywith “D-form polymerase” or “D-polymerase.” For example, “D-Dpo4” refersto D-form Dpo4 polymerase which is in a mirror-image relationship withthe native L-form Dpo4 polymerase.

The polymerase particularly suitable for the present invention includesD-ASFV pol X, D-Dpo4, D-Taq polymerase, and D-Pfu polymerase.

Dpo4 (Sulfolobus solfataricus P2 DNA polymerase IV) is a thermostablepolymerase which can also synthesize DNA at 37° C. Its mismatch rate isbetween 8×10⁻³ to 3×10⁴. It is a polymerase that can replace Taq formulti-cycle PCR reaction. Its amino acid sequence length is within thereach of current chemical synthesis techniques.

Taq polymerase is a thermostable polymerase discovered by Chien andcolleagues in the hot spring microbe Thermus aquaticus in 1976. It canremain active at DNA denaturation temperatures, so it is used in PCRinstead of E. coli polymerase. The optimum temperature for Taq isbetween 75° C. and 80° C. and the half-life at 92.5° C. is about 2hours.

Pfu polymerase is found in Pyrococcus furiosus, and its function inmicroorganisms is to replicate DNA during cell division. It is superiorto Taq in that it has 3 ′-5′ exonuclease activity and can cleave themis-added nucleotides on the extended strand during DNA synthesis. Themismatch rate of commercial Pfu is around 1 in 1.3 million.

In some embodiments, the mirror-image nucleic acid, the mirror-imagenucleic acid template, the mirror-image nucleic acid primer, and themirror-image dNTPs/rNTPs are in L-form, and the mirror-image nucleicacid polymerase is in D-form. In some cases, different types oftemplates, primers, or dNTPs/rNTPs may be mixed in the reaction system(for example, a portion of D-form template primer or dNTPs/rNTPs may bemixed) without causing serious interference with the reaction.

Herein, the nucleic acid replication reaction may be carried out in onlyone cycle or in multiple cycles. This may be determined by personsskilled in the art according to actual needs.

The term “multiple” as used herein refers to at least two. For example,“multiple cycles” refers to 2 or more cycles, such as 3, 4, or 10cycles.

The term “replication” as used herein includes obtaining one or morecopies of a target DNA in the presence of a DNA template and dNTPs; andalso obtaining one or more copies of a target RNA in the presence of aDNA template and rNTPs (this process may also be known as RNA“transcription”).

In the process of nucleic acid replication, the template and the primerare usually DNA. If the target nucleic acid is DNA, dNTPs should beadded to the reaction system; if the target nucleic acid is RNA, rNTPsshould be added to the reaction system.

In some embodiments, the mirror-image nucleic acid is L-DNA, such asL-DNAzyme. In other embodiments, the mirror-image nucleic acid is L-RNA.

In a particularly preferred embodiment, the reaction is a polymerasechain reaction.

The term “PCR” as used herein has a meaning as known in the art andrefers to a polymerase chain reaction.

In a particularly preferred embodiment, the reaction is carried out in abuffer of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT, and 50 mM KCl.

The present invention also provides a method for performing mirror-imagePCR, comprising: carrying out a reaction in the presence of amirror-image nucleic acid polymerase, a mirror-image nucleic acidtemplate, a mirror-image nucleic acid primer, and mirror-imagedNTPs/rNTPs to obtain a mirror-image nucleic acid.

The present invention also provides a method for screening amirror-image nucleic acid molecule, comprising: contacting a library ofrandom mirror-image nucleic acid sequences with a target molecule undera condition that allows binding of the two; obtaining a mirror-imagenucleic acid molecule that binds to the target molecule; and amplifyingthe mirror-image nucleic acid molecule that binds to the target moleculeby mirror-image PCR.

Preferably, the target molecule is immobilized on a solid phase medium,which may be more advantageous for separation and purification.

For example, after the library of random mirror-image nucleic acidsequences is contacted with the target molecule, the mirror-imagenucleic acid molecule that does not bind to the target molecule may beremoved by washing to obtain a mirror-image nucleic acid molecule thatbinds to the target molecule.

The mirror-image nucleic acid molecule can be L-DNA or L-RNA.

Preferably, the mirror-image nucleic acid polymerase used in themirror-image PCR may be D-ASFV pol X, D-Dpo4, D-Taq polymerase or D-Pfupolymerase.

In the present invention, the term “nucleic acid polymerase” should beunderstood broadly and may refer to a wild-type enzyme, and may alsorefer to a functional variant of the enzyme.

The term “functional variant” as used herein refers to a variantcomprising substitution, deletion or addition of one or more (forexample, 1-5, 1-10 or 1-15, in particular, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 15 or even more) amino acids in the amino acidsequence of a wild-type enzyme, and the variant substantially retainsthe biology of the wild-type enzyme. For example, 50%, 60%, 70%, 80% or90% or more of the biological activity of the wild type enzyme isretained. The “functional variant” may be a naturally occurring variant,or an artificial variant, such as a variant obtained by site-directedmutagenesis, or a variant produced by a genetic recombination method.

In a preferred embodiment of the present invention, the mirror-imagenucleic acid polymerase may comprise an affinity tag to facilitatepurification and reuse of the protein, such as a polyhistidine tag(His-Tag or His tag), a polyarginine tag, a glutathione-S-transferasetag, and the like.

For example, a preferred embodiment of the present invention provides afunctional variant of Dpo4 protein, Dpo4-5m, which comprises amino acidmutations at 6 positions and a His6 tag.

In a particularly preferred embodiment, the mirror-image PCR isperformed in a buffer of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT,and 50 mM KCl.

The present invention also provides D-ASFV pol X, the sequence of whichis set forth in SEQ ID NO: 17, wherein except for glycine which is notchiral, all other amino acids are D-form amino acids.

The present invention is further described with reference to theaccompanying drawings and embodiments, which are only for the purpose ofillustrating the present invention and should not be construed aslimiting the present invention.

EXAMPLES Example 1

D-Amino Acid in Mammal

D-amino acids are present in mammals. In 1965, Hoeprich and colleaguesdetected D-Ala in the blood of guinea pig and mouse, which was the firsttime that researchers had discovered D-amino acid in mammals (Corrigan,1969). Until now, D-Ala has been found in the brains and pituitaries ofvarious mammals, and D-Ala excretion has been found in the urine. D-Proand D-Leu were found in seven regions of the mouse brain, indicatingthat D-amino acids have relatively high concentrations in the pituitaryand pineal bodies (Hamase et al., 2001). In addition, D-Ser and D-Alawere also detected in the brains and blood of mammals such as human andmouse (Hashimoto et al., 1993; Hashimoto et al., 1995). D-amino acidswere first thought to be synthesized by microorganisms, plants, andinvertebrates, and recent studies have shown that D-Ser and D-Asp can besynthesized by mammalian tissues. It was found by isotopic labelingexperiments that radioactive L-Ser can be transformed into D-Ser in ratand mouse brains. In 1999, Ser racemase was cloned and purified, whichcan catalyze the conversion of L-Ser to D-Ser in the rat brain (Woloskeret al., 1999). Another major source of D-amino acids in mammals isexogenous foods and microorganisms.

Chemical Synthesis of Mirror-Image Protein

Although the mirror-image amino acids have been confirmed to exist inliving organisms in natural state, the natural state of the mirror-imageamino acids are mainly monomeric, and also exist in short peptidesegments, such as the tetrapeptide side chain amino acidd-Ala-d-Glu-r-l-Lys-d-Ala of Gram-positive bacteria. A mirror-imageprotein consisting of functional mirror-image amino acids in nature hasnot yet been discovered. The translation in the central rule is aprocess in which ribosomes and tRNAs participate and synthesize proteinsaccording to mRNA genetic information. The mirror-image amino acids arenot used as substrates in this process. It is generally believed thatthe transcription, translation and realization of major biologicalfunctions of the genetic information of living organisms depend on thenatural chiral L-amino acids.

Due to the chirality of existing organisms, the mirror-image proteinscannot be obtained by a biological method. At present, the research onmirror-image proteins is achieved by chemical synthesis. Polypeptidesand small proteins can be achieved by solid phase peptide synthesis(SPSS) (Kent, 1988). This method usually yields a segment of about 60amino acids, then the deprotected peptide segments in solution areligated one by one by natural chemical ligation (Dawson and Kent, 2000).The methods of solid phase peptide synthesis and natural chemicalligation extend the range of protein synthesis, and currently cansynthesize a protein of more than 300 amino acids. Synthetic studies ofmirror-image proteins can also be performed by this technique. In 1992,Kent's group synthesized the first mirror-image protease, HIV-1 protease(Milton et al., 1992). Based on the theory that the L and D enantiomershave mutually mirrored structures, scientists often speculate thatmirror-image proteins have functions corresponding to the naturalproteins, and this conjecture was first confirmed in the mirror-imageHIV-1 protease project. L-form and D-form HIV-1 protease have the samemass spectral molecular weight, the same HPLC retention time, and theopposite circular dichroism spectral curves. In terms of activity, theL-form HIV-1 protease can cleave the L-peptide substrate, while theD-form protease can cleave the D-form substrate. In another study, twochiral baczymes (barnase) showed similar properties. Natural L-formbarnase cleaves native RNA, and its activity on mirror-image RNA isabout 4000 times weaker. For mirror-image D-form barnase, the activityon cleaving mirror-image RNA was significantly higher than that onnative RNA (Vinogradov et al., 2015). In 1995, L-form and D-form4-Oxalocrotonate tautomerase were chemically synthesized, and the twoenantiomers showed the same reaction efficiency in catalyzing theisomerization of an achiral substrate 2-hydroxymuconic acid.Isotopically labeled hydrogen at the carbon atom of the catalytic siteindicated that two chiral isomerases act on different sides of thecarbon atom (Fitzgerald et al., 1995). The above studies consistentlyshow that the mirror-image protease and the natural chiral protease havethe same activity, but act on different isomeric positions.

In 2014, M. S. Kay's group synthesized the longest protein, DapA of 312amino acids. DapA is a protein that relies on a molecular chaperoneGroEL/ES and can be only folded into a functional conformation uponexpression with the assistance of the molecular chaperone. GroEL/ES canfold both D- and L-chiral DapAs, but the folding efficiency for nativeL-DapA is higher than that for mirror-image DapA (Weinstock et al.,2014).

Study on Chemical Synthesis of Mirror-Image Nucleic Acid

Studies on the hypothesis of the early origin of life suggest thatnon-enzymatically catalyzed, template-directed RNA amplificationreactions can be performed in a single chiral system. However, if thereare two chiral RNA monomers in the system, the prolonged reaction willbe prevented by the addition of its mirror-image monomer (Joyce et al.,1984). This poses a serious challenge to the theory that life originatesfrom naturally occurring RNA. To explain this theoretical problem, Joyceand colleagues obtained an RNA polymerase ribozyme by in vitroscreening, which consists of 83 ribonucleosides and can catalyzeenantiomeric chiral RNA polymerization (Sczepanski and Joyce, 2014). Themirror-image RNA in this study was obtained by chemical synthesis. Itcould generate a full-length RNA polymerase ribozyme opposite to its ownchirality by ligating 11 oligonucleotide fragments, and amplification ofthe two RNAs can be performed without interference in the same reactionsystem. This provided a theoretical possibility for the coexistence oftwo chiral RNA molecules and amplification of them by RNA polymeraseribozyme in the early stage of life.

In 2013, Barciszewski's group reported for the first time acatalytically active mirror-image nucleotidase designed according to theexisting natural nucleotide sequence, which functioned to catalyze thecleavage of the mirror-image L-nucleotide molecule (Wyszko et al.,2013). The mirror-image nucleotidase can achieve a cleavage function invivo. In the experiment, it was confirmed that the mirror-imagenucleotidase was not easily degraded in serum, and also had thecharacteristics of being non-toxic and not causing an immune reaction,which made it an ideal drug molecule.

Study on Mirror-Image Nucleic Acid Aptamer

DNA/RNA was first thought to be a carrying tool for genetic informationand also thought to be much simpler than the structure of a protein. Butactually DNA/RNA can also be folded into a tertiary structure, which hasa series of potential physiological functions. As early as in 1990,researchers discovered that RNA structure can specifically bind to asmall molecule substrate. These RNA structures, like antibodies, bindselectively to the substrate and have a high affinity. These RNAstructures that can bind to a particular substrate are referred to asaptamers. Later, DNA aptamers were also discovered by researchers.

In vitro screening technique utilize a library of random DNA/RNAsequences to find the nucleic acid aptamer that binds to a specifictarget molecule. In vitro screening must first have a library of randomsequences, either DNA or RNA. The library typically contains randomsequences of 30 to 80 nucleotides with two primer regions on both sidesto facilitate PCR amplification. Then, multiple cycles of the screeningprocess are performed, the target small molecule substrate isimmobilized on a matrix, and the library of random sequences is added tothe substrate. After washing, the unbound DNA or RNA molecule flowsthrough the immobilized substrate, and the sequence with the bindingability that is screened will remain on top. The special sequence isthen eluted for PCR amplification, and after multiple cycles ofenrichment and screening, one or several nucleotide sequences thatspecifically bind to the substrate can be obtained.

Like the natural nucleic acid aptamer, the mirror-image nucleic acidsequence has a specific secondary and tertiary structure according toits sequence, and can bind the target molecule in close and highspecificity. If a mirror-image target molecule is screened by an invitro screening strategy and then a mirror-image nucleotide sequence issynthesized, a mirror-image nucleotide molecule capable of binding to anatural target can be obtained. The researchers obtained a mirror-imageL-RNA aptamer that binds D-adenosine and L-arginine by in vitroscreening (Klusmann et al., 1996; Nolte et al., 1996). D. P. Bartelobtained an L-DNA aptamer that binds vasopressin in 1997 (Williams etal., 1997). The mirror-image nucleotide molecule has the advantages ofbeing stable and not easily degraded in the body, non-toxic, and notcausing an immune reaction, has a relatively low production cost, andhas a good application prospect as a drug molecule.

Research Purposes

No research can give a clear conclusion whether mirror-imagebiomolecules can realize the replication and transcription of geneticinformation, and whether mirror-image molecules have the theoreticalpossibility of composing life in evolution. Although mirror-imageproteins have been synthesized and the activities of mirror-imageproteins have been verified and their properties have been compared withnative proteins by research groups, these studies usually only explainthe properties of mirror-image proteins from the perspective of chemicalsynthesis.

Our research aimed to design and synthesize a polymerase-basedmirror-image replication and transcription system. The significance ofthis system has the following three aspects:

I. The mirror-image replication and transcription system can realize theamplification of mirror-image DNA and RNA by polymerase, indicating thatthe mirror-image polymerase can catalyze the synthesis of DNA and RNAlike natural chiral polymerase, and proving that the mirror-imagebiomolecule has effective biological activity;

II. The mirror-image replication and transcription system implements twokey steps in the mirror-image center rule, laying a groundbreakingfoundation for the synthesis of mirror-image primitive cells;

III. At present, the natural nucleic acid molecule obtained by in vitroscreening technique as a drug has a serious defect of being easilyhydrolyzed in the body. In order to avoid this problem, a special methodis needed for screening of mirror-image nucleic acid. The existing invitro screening technology screens the mirror-image target by a naturalrandom library to obtain an effective nucleic acid sequence, and thenchemically synthesizes the mirror-image nucleic acid molecule, so thatthe obtained mirror-image nucleic acid molecule can bind to a naturaltarget, that is, a potential mirror-image drug. However, the limitationof this method is that many common drug targets in living organisms areproteins of more than 300 amino acids, and it is impossible tosynthesize the mirror-image targets by a chemical method. If the naturaltarget molecule and the mirror-image random library can be directly usedfor mirror-image in vitro screening, the universality of this techniquewill be greatly improved, and drug molecules will be screened for a widerange of diseases. There are no technical difficulties for the naturaldrug target and the mirror-image random library, but the bottleneck ofmirror-image drug screening is that mirror-image PCR cannot be realized.Our mirror-image replication and transcription system can achievemirror-image PCR. Although PCR efficiency needs further optimization andimprovement, it still provides a theoretical and practical basis formirror-image drug screening.

Experimental Materials and Experimental Methods

Experimental Drugs and Reagents

Q5 DNA polymerase NEB NdeI NEB BamHI NEB Trans 5α clone competent cellsTransGen Biotech BL21(DE3)pLysS expression competent cells TransGenBiotech EasyTaq TransGen Biotech Trans 2K DNA marker TransGen Biotech T4DNA ligase TransGen Biotech Plasmid miniprep kit Aidlab IPTG Aidlab GelExtraction Kit QIAgen Agarose Biowest Pre-stained protein marker Bio-Rad40% Acrylamide Sangon Urea Sangon SYBRGold stain solution InvitrogenCoomassie Brilliant Blue Fast Stain solution Tiangen Biotech (Beijing)Ni-NTA beads Institute of Process Engineering, CAS Imidazole SangonD-amino acids CSBio (Shanghai) L-amino acids GL Biochem (Shanghai)2-(7-azobenzotriazole)-N,N,N′,N′-tetramethyluron GL Biochem (Shanghai)hexafluorophosphate 1-hydroxy-7-azobenzotriazole GL Biochem (Shanghai)N-hydroxy-7-azobenzotriazole GL Biochem (Shanghai)1-hydroxy-benzo-triazole GL Biochem (Shanghai)N,N′-dicyclohexylcarbimide Alfa Aesar 1,2-ethanedithiol and4-mercaptophenylacetic acid Alfa Aesar Acetonitrile (HPLC grade) J.T.Baker Tris(hydroxymethyl)methylamine hydrochloride Sinopharm (Tris-HCl)Potassium chloride Sinopharm Magnesium acetate tetrahydrate SinopharmEthylenediaminetetraacetic acid Sinopharm Sodium dihydrogen phosphateSinopharm Glycerin Sinopharm Guanidine hydrochloride Sinopharm Anhydrousether Sinopharm 85% Hydrazine Sinopharm Reduced glutathione J&KScientific Anisole sulfide J&K Scientific Trifluoroacetate J&KScientific Triisopropylsilane Beijing Ouhe TechnologyN,N-diisopropylethylamine Beijing Ouhe Technology Sodium hydroxideBeijing Chemical Industry Group N,N-dimethylformamide Beijing ChemicalIndustry Group Dichloromethane Beijing Chemical Industry Group Sodiumnitrite Beijing Chemical Industry Group 2-chlorotriphenyl chloride resinNankai Hecheng (Tianjin) β-mercaptoethanol Zhongketuozhan (beijing)L-DNA Chemgenes L-dNTPs Chemgenes L-NTPs Chemgenes D-DNA primer Tsingke(Beijing) pEASY T3 Kit TransGen Biotech RNase A Fermentas PAGE DNAPurification Kit Tiandz Phenol chloroform Solarbio Glycogen Aidlab RNaseinhibitor TaKaRaExperimental Equipments

Nucleic acid electrophoresis tank Beijing JUNYI PAGE electrophoresistank Beijing JUNYI Benchtop thermostatic oscillators Huamei BiochemicalInstrument SDS-PAGE electrophoresis tank Bio-Rad Electrophoresisapparatus Bio-Rad Gel imaging system Bio-Rad Centrifuge5424 EppendorfSpectrophotometer Eppendorf PCR instrument AB Milli-Q Pure WaterInstrument MilliPore AKTA Protein Purification System GE QIAcube QIAgenNucleic Acid Sequences

Name Sequence D-/L-primer12 5′-ACTACGAACGCG (SEQ ID NO: 1)D-/L-FAM-primer12 5′-FAM-ACTACGAACGCG (SEQ ID NO: 2) D-/L-template185′-CTCAGTCGCGTTCGTAGT (SEQ ID NO: 3) D-/L-DNAzymeTemplate5′-TGTACAGCCACTTCAACTAATTGCTCAACTATGGCTGTAGCACCCGCGTTCGTAGTATGCAATGCA (SEQ ID NO: 4) Cy5-primer205′-Cy5-AGTGCGATACTACGAACGCG (SEQ ID NO: 5) template265′-CTCAGTCGCGTTCGTAGTATCGCACT (SEQ ID NO: 6) template18A5-CTCAGACGCGTTCGTAGT (SEQ ID NO: 7) template18C 5′-CTCAGCCGCGTTCGTAGT (SEQ ID NO: 8) template18G5′-CTCATGCGCGTTCGTAGT (SEQ ID NO: 9) L-primer155′-GATCACAGTGAGTAC (SEQ ID NO: 10) L-template215′-CTATTGTACTCACTGTGATC (SEQ ID NO: 11) DNAzyme marker5′-FAM-ACTACGAACGCGGGTGCTACAGCCATAGTTGAGCAATTAGTTGAAGTGGCTGTACA (SEQ ID NO: 12) L-template275′-CGCGCTGTTATAGGGATACGGCAAAAA (SEQ ID NO: 13) L-primer 115′-CGCGCTGTTAT (SEQ ID NO: 14) L-FAM-primer115′-FAM-CGCGCTGTTAT (SEQ ID NO: 15) L-reverse115′-GCCGTATCCCT (SEQ ID NO: 16)

The above sequences without D-/L-tags refer to D-DNA.

ASFV Pol X Protein and DNA Sequence

Protein Sequence

(SEQ ID NO: 17)   1mltliqgkki vnhlrsrlaf eyngqlikil sknivavgsl rreekmlndv dlliivpekk  61llkhvlpnir ikglsfsvkv cgerkcvlfi ewekktyqld lftalaeekp yaifhftgpv 121syliriraal kkknyklnqy glfknqtlvp lkittekeli kelgftyrip kkrlDNA Sequence

(SEQ ID NO: 18) ATGTTAACGCTTATTCAAGGAAAAAAAATTGTAAATCACTTACGTTCCCGACTTGCGTTTGAATATAATGGACAACTTATAAAAATTTTATCAAAAAACATCGTTGCTGTTGGTAGTTTAAGACGCGAAGAGAAAATGCTTAATGACGTGGATCTTCTTATTATTGTTCCAGAAAAAAAACTTTTAAAACACGTCCTGCCCAACATTCGCATAAAGGGTCTTTCTTTTTCTGTAAAAGTCTGCGGAGAACGAAAGTGTGTACTTTTTATTGAATGGGAAAAAAAGACGTATCAACTTGATCTTTTTACGGCTTTAGCCGAGGAAAAACCATACGCAATATTTCATTTTACGGGTCCCGTTTCTTATCTAATAAGAATTCGAGCCGCGTTAAAAAAAAAGAATTATAAGCTAAATCAGTATGGATTATTTAAAAATCAAACTTTAGTACCTCTAAAAATCACTACTGAAAAAGAACTTATTAAAGAATTAGGATTTACGTATCGCATACCTAAGAAACGTTTATAAExperimental MethodsChemical Synthesis of ASFV Pol X

The amino acid sequence of D-ASFV pol X was divided into three segments,and a natural ligation method from the C-terminus to the N-terminus wasadopted. The synthesis of each polypeptide segment used a solid phasepeptide synthesis method (Fmoc-SPPS) based on a strategy of using9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group. 2-C1-trityl-C1resin (2CTC, degree of substitution 0.5 mmol/g) was used to synthesizesegment 1, while hydrazine-substituted 2CTC resin was used to synthesizesegment 2 and segment 6. In the synthesis of the polypeptide segment,the resin was first swollen in a mixture of dichloromethane (DCM) andN,N-dimethylformamide (DMF) for half an hour, and then the solvent wasremoved. Subsequently, for the segment 1, a solution of 4 eq. aminoacids and 8 eq. N,N-diisopropylethylamine in 5 ml of DMF was added tothe reaction tube, and the reaction was carried out for 12 hours in athermostat shaker at 30° C., after which 200 μL of methanol was added toblock unreacted active chlorine. For the segment 2 and segment 6, asolution of 4 eq. amino acids, 3.8 eq.2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluoride phosphate(HATU), 3.8 eq. benzotriazole (HOAT) and 8 eq. N,N-diisopropylethylaminein 5 ml of DMF was added to the reaction tube, and the reaction wascarried out for 1 hour in a thermostat shaker at 30° C. During thesynthesis of the polypeptide segment, the method of removing the Fmocprotecting group comprised the use of a DMF solution containing 20%piperidine for soaking twice, one for 5 minutes, and the other for 10minutes. From the second amino acid to the end, the condensation systemused HATU/HOAT/DIEA. After the peptide segment was synthesized, it wasimmersed for 3 hours with the cleavage reagent K (Trifluoroaceticacid/phenol/water/thioanisole/ethanedithiol=82.5/5/5/5/2.5), then thesystem was concentrated by removing the trifluoroacetic acid withhigh-purity nitrogen gas, then diethyl ether was added to precipitatethe polypeptide, and finally, the solid precipitate was collected bycentrifugation, and the target polypeptide segment was separated andpurified using a semi-preparative grade reversed-phase high performanceliquid chromatography (RP-HPLC).

The natural chemical ligation of the polypeptide segments was carriedout as follows. The hydrazide group-containing polypeptide segment wasfirst dissolved in a buffer (6 M guanidine hydrochloride, 200 mMdisodium hydrogen phosphate, pH 3.0). In an ice salt bath, a 15 eq.NaNO₂ solution was added to the reaction solution, and the reaction wascarried out for 20 minutes. Subsequently, a buffer solution mixed with40 eq. 4-mercaptophenylacetic acid (MPAA), an equivalent of N-terminalcysteine, pH 7.0 was added. After stirring uniformly, the pH of thesystem was adjusted to 7.0 and the reaction was carried out for 12hours. After the reaction was completed, the system concentration wasdiluted by 2-fold with addition of 80 mM of trichloroethyl phosphate(TCEP) buffer. The target product was finally isolated usingsemi-preparative grade RP-HPLC.

The desulfurization reaction of the polypeptide was carried out asfollows. First, 1 μmol of the polypeptide segment 3 was dissolved in 2.5ml of 200 mM TCEP buffer solution (6 M guanidine hydrochloride, 0.2 Mdisodium hydrogen phosphate, pH=6.9), then 50 μmol VA-044 and 100 μmolreduced glutathione were added, and the solution was reacted at 37° C.for 12 hours. Finally, the desulfurization product 4 was isolated andpurified by semi-preparative grade RP-HPLC.

The acetamidomethyl (Acm) protecting group of the Cys86 side chain wasremoved as follows. 0.5 μmol of the polypeptide segment 4 was dissolvedin 1 ml of 50% aqueous acetic acid. Then 5 mg of silver acetate wasadded and stirred at 30° C. overnight. Then 2.5 mmol of mercaptoethanolwas added and the system was diluted by 2-fold with 6 M aqueousguanidine hydrochloride solution. The precipitate was removed bycentrifugation, and the supernatant was separated by RP-HPLC to obtainthe target product 5.

Folding Renaturation of ASFV Pol X

The folding renaturation of D-ASFV pol X was carried out as follows. 5mg of D-ASFV pol X was dissolved in 10 ml of 6 M guanidine hydrochloridesolution, and the solution was placed in a 3K Da dialysis bag. Thedialysis bag was then immersed in a buffer system containing 4 Mguanidine hydrochloride (50 mM Tris-HCl, 40 mM KCl, 6 mM magnesiumacetate, 0.01 M EDTA and 16% glycerol) for 10 hours, then the guanidinehydrochloride concentration was gradually reduced to 2 M, 1 M and 0 M.The dialysis bag was immersed in each concentration of guanidinehydrochloride solution for 10 hours. Using circular dichroism and massspectrometry, it was confirmed that D-ASFV pol X was correctly foldedand a disulfide bond was formed by air oxidation between D-Cys81 andD-Cys86.

Natural and Mirror-Image DNA, RNA Polymerase Reaction Method

DNA polymerization method: a polymerase reaction buffer of 50 mMTris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT, and 50 mM KCl was formulated.0.7 μg of ASFV pol X, 2.5 μM primer, 2.5 μM template and four kinds of0.4 mM dNTPs were added to a 10 μl reaction system. The reaction systemwas placed at 37° C. for 4 hours, and the reaction was terminated byadding 1 μl of 0.5 M EDTA. The reaction yielded a DNA fragmentcomplementary to the template.

RNA polymerization method: a polymerase reaction buffer of 50 mMTris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT, and 50 mM KCl was formulated.0.7 μg of ASFV pol X, 2.5 μM primer, 2.5 μM template and four kinds of0.4 mM rNTPs were added to a 10 μl reaction system. The reaction systemwas placed at 37° C. for 60 hours, and the reaction was terminated byadding 1 μl of 0.5 M EDTA. The reaction yielded a complex of primer DNAand RNA.

Method for Recovering DNA/RNA Fragment by PAGE Gel

The amplified product was isolated and purified by a PAGE gel recoverymethod. First, a loading buffer containing xylene cyanide andbromophenol blue was added to the system in which the reaction had beenterminated, and electrophoresis was carried out on a modifiedpolyacrylamide gel of a suitable concentration, and the gel was removedafter the electrophoresis was completed. DNA/RNA staining was performedwith ethidium bromide. The target fragment of the DNA/RNA with desiredsize was cut under UV light, and the impurity band and the blank areawere discarded. The cut gel piece should be as small as possible. Thegel was then placed in TE buffer and mixed upside down overnight. Thesupernatant was carefully aspirated, and 1/10 volume of sodium acetate(3 mol/L, pH=5.2) was added to the DNA solution and mixed well to afinal concentration of 0.3 mol/L. After adding 2 times volumes ofpre-cooled ethanol with ice for mixing, it was thoroughly mixed againand placed at −20° C. for 30 minutes. The mixture was centrifuged at12,000 g for 10 minutes, then the supernatant was carefully removed andall droplets on the tube wall were aspirate. The open lid EP tube wasplaced on the bench at room temperature to allow the residual liquid toevaporate to dryness. By adding an appropriate amount of ddH₂O todissolve the DNA/RNA, high purity enzymatically amplified L-DNA/RNAfragment can be obtained.

Mirror-Image DNAzyme Reaction Method

A DNAzyme sequence was amplified using a 100 μl reaction system, whichis a single-stranded DNA short strand with self-splicing activity. Thereaction system was: 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 1 mM DTT, and50 mM KCl, 28.9 μg of D-ASFV pol X, 5 μM primer12 primer, 5 μMDNAzymeTemplate and 1.6 mM dNTPs. The reaction was carried out at 37° C.for 36 hours. Next, the reaction product was added to the loadingbuffer, and the band was separated on a 12% PAGE gel using 300 V for 3hours. In order to facilitate the separation by the gel and the recoveryof the full-length reaction product, the single-stranded DNAzyme, theDNAzymeTemplate was designed to be 10 nucleotides longer than thefull-length product. After the gel plate was removed, the full-lengthproduct sequence was developed and excised. The gel piece was treated asdescribed above, diffused overnight and precipitated by ethanol torecover the DNA. The precipitated product was dissolved in a buffer of1:50 mM HEPES, pH 7.0 and 100 mM NaCl, and heated at 90° C. for 2minutes, and then cooled on ice for 5 minutes. An equal volume of abuffer of 2:50 mM HEPES, pH 7.0, 100 mM NaCl, 4 mM ZnCl₂ or 40 mM MgCl₂was then added to initiate the reaction. The reaction was carried out at37° C. for 36 hours. Finally, the reaction was terminated with EDTA. Thesample was separated and developed on a 12% PAGE gel.

Mirror-Image Multi-Cycle Polymerase Reaction Method

A polymerase reaction buffer of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1mM DTT, and 50 mM KCl was formulated. 2.672 μg of D-ASFV pol X, 2.5 μML-FAM-primer, 2.5 μM L-template and four kinds of 0.4 mM L-dNTPs wereadded to the reaction system. The reaction system was placed at 37° C.for 4 hours, and the reaction was terminated by adding 1 μl of 0.5 MEDTA. Since ASFV pol X has a strong ability to bind DNA and it cannot bedissociated even under the condition of heat denaturation at 95° C., thepolymerase in the previous cycle of reaction was removed by phenolchloroform extraction. In the second cycle of reaction, the reactionsystem was heated to 95° C. for 30 seconds and then cooled to roomtemperature. Further, 0.334 μg of D-ASFV pol X was added to the reactionsystem, and the reaction system was allowed to stand at 37° C. for 20hours. The third cycle was carried out in the same way. The reactionproduct was separated on 8 M urea denatured 20% PAGE gel and imagedusing the Typhoon Trio+ system. Quantification of the electrophoresisband was performed using ImageQuant software.

Digestion of Natural RNA Polymer Product by RNase A

The L-ASFV pol X RNA reaction system (see 2.2.3) was reacted at 37° C.for 36 hours, and then was heated to 75° C. for 10 minutes to inactivateASFV pol X and RNase inhibitor.

1 μg/μl, 0.1 μg/μl, and 0.01 μg/μl of RNase A were respectively added toeach of the three experiments and incubated at 23° C. for 10 minutes.The reaction was terminated by the addition of 20 units of RNaseinhibitor and a loading buffer was added. The reaction product wasseparated on 8 M urea denatured 20% PAGE gel and imaged using theTyphoon Trio+ system.

Design of Genetic Information Replication and Transcription System forMirror-Image Chirality

Overview

In the organism of the earth, the amino acids constituting the proteinsare almost L-form except for glycine which has no charility, and theriboses in the nucleic acids are all D type. Proteins and nucleic acidsare characterized by chiral unitarity. The erroneous addition of one orseveral mirror-image amino acids to a natural protein may alter thesecondary structure of the protein or even lose its biological activity(Krause et al., 2000). The organism has a strict chiral unitarity.Although researchers have not been able to find clear evidence of whythe mirror-image chirality is lost in evolution, we can still study theproperties of mirror-image proteins and nucleic acids by chemicalsynthesis, and try to construct the biological primitives required forthe mirror-image organism of the cells. As the core of the mirror-imageorganism, the research focuses on trying to construct key steps in themirror-image center rule, DNA replication and RNA transcription.

Design of Mirror-Image DNA Replication System

In natural living organisms, DNA replication requires a long DNA strandas a template, a short DNA strand as a primer, a DNA polymerase, fourkinds molecules of dATP, dCTP, dTTP, and dGTP, and suitable solutionconditions such as suitable pH and Mg²⁺ ion. With reference to thenatural life system, the components of a mirror-image DNA replicationsystem were designed, comprising 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 1mM DTT, and 50 mM KCl, with addition of mirror-image protein and nucleicacid molecule, D-polymerase, L-DNA template, L-DNA primer and fourL-dNTPs. The mirror-image protein and nucleic acid molecule do not existin natural living organisms. For chemical synthesis processes, there isno difference in the chemical processes required to synthesize a chiralmolecule and an enantio chiral molecule. So, it was determined to obtaina mirror-image D-polymerase and

L-DNA by Chemical Means.

The chemical synthesis of protein is mainly through the solid phasesynthesis of peptides of about 60 amino acids, and the deprotectedpeptides are ligated one by one through natural chemical ligation insolution. The upper limit of the protein that can currently besynthesized is approximately 350 amino acids depending on the sequence.The size of E. coli polymerase I is 928 amino acids, the commonly usedtaq polymerase has 832 amino acids, and the T4 DNA ligase, which isgenerally considered to have a simpler function of the polymerase, alsohas 487 amino acids. These enzymes are beyond our synthesis range.Polymerase ASFV pol X, African swine fever virus polymerase X, of 174amino acids was selected through literature search.

ASFV encodes two DNA polymerases, one is an eukaryotic type DNApolymerase of family B for replication of the viral genome; and theother is a DNA polymerase of family X, named ASFV pol X (Oliveros etal., 1997). ASFV Pol X is the smallest DNA polymerase found today(Showalter and Tsai, 2001), consisting of 174 amino acids and having asize of 20 kDa. ASFV Pol X is a template-dependent polymerase with verylow fidelity, lacks 3′-5′ exonuclease activity, and has poor recognitionability for dideoxynucleotides (Oliveros et al., 1997).

By aligning the gene sequence of ASFV Pol X with other representativepolymerases of family X, it can be seen that ASFV Pol X and humanbovine, murine TdT, human, murine pol β, etc. have a certainrelationship in structure and function. The three-dimensional structureof ASFV Pol X has been solved by NMR. Unlike other polymerases, Pol Xhas only one palm structure and one C-terminal domain (Maciejewski etal., 2001). In eukaryotic Polβ, there is generally an N-terminal domainresponsible for DNA binding. Pol X does not have this key domain, butits ability to bind DNA is stronger than that of Polβ.

In addition, ASFV Pol X can bind to the intermediate of a singlenucleotide excision repair (BER) and efficiently repair individualnucleotide gap (Showalter et al., 2001). In the BER step, Pol X repairsthe gap and is easy to introduce mutation. In the final step, a newsynthetic strand with a mismatch is ligated into the genome by afault-tolerant ligase encoded by ASFV. Pol X introduces new mutationwhen repairing the genome, which helps the virus to mutate in order tosurvive in an environment with stress.

Chemical Synthesis of ASFV Pol X Polymerase

The chemical total synthesis method of D-ASFV PolX uses the solid phasepeptide synthesis technology (SPPS) proposed by Merrifield in 1963,which is currently the most effective method for peptide synthesis.Depending on the protection strategy, there are mainly two methods ofadopting tert-butoxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc).In this project, it was proposed to use a Fmoc-solid phase synthesistechnique to ligate D-form amino acids into a polypeptide.

Although in theory the yield of each step of the SPPS condensationreaction can reach 99%, in practice Fmoc-solid phase synthesistechnology can usually only synthesize peptide chains of less than 50amino acids. For this reason, synthesis of protein often needs to ligatethe polypeptide segments, in other words, a target protein molecule isdivided into several segments such that each segment has less than 50amino acids, and then each segment is ligated by a highly efficientchemical reaction to obtain the target protein molecule. Therefore, inthis project, it was proposed to divide the polypeptide chain of thetarget D-ASFV protein molecule into 4 segments, and using a convergencemethod of two-two combination, the full-length amino acid sequence ofthe target protein was obtained by a modified one-pot natural chemicalligation (NCl), protein hydrazide ligation method.

Since the site of the hydrazide ligation must utilize cysteine, it wasintended to mutate the alanine at position 36 and position 129 tocysteine. After completion of the ligation reaction, a desulfurizationreaction was carried out to reduce cysteine to alanine (Qian andDanishefsky, 2007). At the same time, in order to avoid the occurrenceof side reaction during the ligation reaction, it was necessary to carryout side chain sulfhydryl protection for the cysteine at positions 81and 86, and then to carry out the removal after the completion of theligation reaction. It was intended to use acetamidomethyl (Acm) as athiol protecting group during the reaction (Liu et al., 2012). Thespecific synthetic route and steps for synthesizing D-ASFV pol X areshown in FIG. 1.

For verifying the feasibility of the synthetic route, and also acost-saving consideration, the synthesis of L-ASFV pol X was firstcarried out. Thereafter, D-ASFV pol X was synthesized in the samemanner.

Renaturalation and Analytical Detection of Chemical ASFV Pol XPolymerase

After obtaining the full-length L- and D-ASFV pol X, they were subjectedto reverse-phase chromatography (RP-HPLC) purification using a Vydac C18(4.6×250 mm) liquid chromatography column with TFA and separated with a20% to 70% acetonitrile gradient (FIG. 2). A strong absorption of themain peak and a weak impurity peak were obtained. The molecular weightof the synthetic full-length product of D-ASFV pol X determined byelectrospray ionization mass spectrometry (ESI-MS) was 20317.0 Da, andthe theoretical value was 20316.0 Da.

The synthesized D-ASFV pol X was renatured by denaturation-dialysismethod. The lyophilized polymerase dry powder was first added to 10 mlof a dialysate containing 6 M guanidine hydrochloride, and the dialysatecontained 50 mM Tris-HCl, pH 7.4, 40 mM KCl, 6 mM (AcO)₂Mg, 0.01 M EDTAand 16% glycerol. During the dialysis process, the concentration ofguanidine hydrochloride in the dialysate was continuously lowered from 4M, 2 M to 0, and the dialysis was stirred at 4° C. for 10 hours eachtime. A disulfide bond was formed between D-Cys81 and D-Cys86 by airoxidation. The E. coli expressed, the synthesized L- and the syntheticD-ASFV pol X were also compared by SDS-PAGE, and their band positionswere consistent (FIG. 3). We also observed a small number of peptidesegments that were not completely ligated. We compared the L- and D-ASFVpol X by circular dichroism spectroscopy (CD). Since D-ASFV pol X cannotbe degraded by common proteases (such as trypsin and pepsin, theexperimental results are not listed) and cannot be sequenced, thesequence of L-ASFV pol X was analyzed by mass spectrometry only, and100% of the pol X sequence was covered in the test results (Table 3.1).

TABLE 3.1Chemically synthesized L-ASFV pol X mass spectrometry sequencingpeptide segment sequences MH+ ΔM RT Segment sequence XCorr Charge [Da][ppm] [min] EEKmLNDVDLLIIVPEK 4.86360168 2 2014.09123  6.6768684443.7154 HVLPNIRIKGLSFSVKVcG 2.49292898 3 2409.36637  4.78211214 44.34 ERKcVLFIEWEK 3.71132851 2 1351.71477  5.0557844 38.6446 KKTYQLDLFTALAE3.88172317 2 1640.89983  6.31761428 43.7574 KLLKHVLPNIR 3.55191755 21330.87224  3.02709865 31.6344 KTYQLDLFTALAEEKPYAI 6.0060935 33631.95664 10.0763472 54.9092 FHFTGPVSYLIR LNQYGLFKNQTLVPLKITT2.74559188 5 4533.6656  7.29527526 54.3708 EKELIKELGFTYRIPKKRLmLTLIQGKKIVNHLRSRLA 2.66363716 3 3299.90256  7.61680303 49.6173FEYNGQLIK NYKLNQYGLFK 3.87206316 2 1387.74187  3.58682542 33.5826SRLAFEYNGQLIKILSKNI 2.5462563 5 3431.98255  9.8659898 54.0116VAVGSLRREEKDetection of Mirror-Image DNA

After the successful synthesis of D-ASFV pol X polymerase, themirror-image nucleic acid was still missing in the mirror-image geneticinformation replication system. The mirror-image DNA fragments such asL-primer12 and L-template18 (see 2.1.3 for the sequence) and fourL-dNTPs were purchased from Chemgenes, USA.

BRIEF SUMMARY

A system for mirror-image DNA replication was designed, including amirror-image polymerase, a mirror-image DNA, four mirror-image dNTPs,and appropriate buffer conditions. The mirror-image DNA and four dNTPswere obtained from Chemegenes. Since the mirror-image protein could notbe obtained by a biological method, ASFV pol X polymerase of 174 aminoacids was found, and a synthetic route was designed and L- and D-ASFVpol X polymerase were synthesized. The protein was folded into thecorrect conformation by guanidine hydrochloride denaturation-dialysisrenaturation. After detection by mass spectrometry and SDS-PAGE, it wasverified that the size of the chemically synthesized protein wasconsistent with the native protein. Further, by CD detection, theabsorption spectra of the native and mirror-image polymerases wereobserved to be symmetric. The correctness of the chemically synthesizedprotein sequence was verified by sequencing the synthetic L-ASFV pol X.Thus, various components of the mirror-image DNA replication system hadbeen obtained, and conditions for subsequent studies on the mirror-imagepolymerase reaction had been complete.

Functional Study of Mirror-Image Chiral Genetic Information Replicationand Transcription System

Overview

So far, there is no research on the replication and transcription ofmirror-image DNA. For the synthetic mirror-image polymerase, first itneeds to verify the possibility of interaction between the mirror-imageprotein and DNA, the chiral specificity of the mirror-image polymerase,and whether the replicated DNA is biologically active. Meanwhile,attempts have been made to achieve multi-cycle amplification of themirror-image DNA, which can be used in research to obtain moremirror-image nucleic acid molecules in the future. Since some enzymes inthe X polymerase family to which ASFV pol X belongs can utilize NTP andtemplate complementary amplification to obtain RNA molecule, it was alsoattempted to try template-based transcription based on the mirror-imageDNA replication system.

DNA Extension Catalyzed by Mirror-Image Polymerase

A mirror-image DNA replication system has been designed and the majorcomponents of the system have been obtained by chemical synthesis (FIG.5a ). In an achiral pH 7.5 Tris-HCl buffer, 20 mM Mg²⁺ was added, 0.7 μgof L-ASFV pol X polymerase, 2.5 μM D-template18, 2.5 μM D-primer12 and0.2 mM D-dNTPs were added to the native system; the same concentrationof D-ASFV pol X polymerase as well as D-primer, template and dNTPs wereadded to the mirror-image system. The reaction systems were allowed tostand at 37° C. for 4 hours. A fluorescein phosphoramidate (FAM) labelwas added to 5′ ends of the both L-form and D-form primers, while thetemplate was not modified. Thus, the primer band could be seen byexcitation with 488 nm light without dyeing. In both systems, bothnative and mirror-image 12 nt primers extended to 18 nt (FIG. 5b ). Onthe 8M urea denatured 20% PAGE gel, the position of each base from 12-18nt can be clearly distinguished. 0 h was used as a control group, theprimer length was 12 nt, and 4 h was a fully extended end product with alength of 18 nt. According to the template sequence, it was speculatedthat the extended 6 bases were ACTGAG, indicating that four kinds ofdNTPs can be used as substrates to synthesize DNA strands in themirror-image system. This conclusion still required subsequentcomplementary pairing verification. It was further desirable to verifythat the mirror-image extension product has the correct mirror-imagenucleoside added. Since the current generation of sequencing methodsstill relies on polymerases and natural dNTP derivatives, this systemcannot be used for L-DNA sequencing. An attempt was made to verify themass of the 18 nt extension product by mass spectrometry. The molecularweight of the full-length mirror-image extension product was 5516.9 Da(theoretical value of 5517.6 Da) by ESI-MS. The full-length product of18 nt was considered to have the correct mass within the error range. Inaddition, it was desirable to understand whether the mirror-imageamplification system has limitations and whether it can be applied todifferent primers and template sequences. In another set of experiments,a 15 nt primer and a 21 nt template (15 nt primer without FAM labeling,PAGE gel stained with Sybr Gold) were used, and the phenomenons ofprimer reduction, appearance of intermediate band, and an increase inthe amount of full-length DNA could still be observed, indicating thatthe mirror-image DNA extension can be used for different DNA sequences(FIG. 5d ).

Mirror-Image DNA Replication System

An attempt was made to perform a multi-cycle PCR amplification of themirror-image DNA replication system. Although ASFV pol X is not athermostable enzyme, multiple cycles of amplification can be performedby adding enzyme per cycle. The binding of ASFV pol X to DNA blocked theamplification of the next cycle, so phenol chloroform extraction wasperformed at the end of each cycle to remove the protein, followed bythe addition of fresh ASFV pol X. Extraction per cycle resulted in alarge DNA loss (recovery efficiency of approximately 40%) and only 3cycles of amplification were preformed. Since the number of cycles wassmall and it was difficult to detect an increase in DNA product, aFAM-labeled primer was used, and it can only use the full-lengthextension product produced in the first cycle for the second round ofamplification (FIG. 6). FAM-labeled primer11, unlabeled reverse11, andtemplate27 were used in the reaction, and the full-length product was 22nt (see 2.1.3 for the sequence). By quantification of the FAM-labeledfull-length product, a 2.3-fold amplification was detected from thesecond to the third cycle, and the theoretical amplification value was 3(FIG. 7). The results theoretically confirmed the feasibility ofmulti-cycle amplification of DNA in the mirror-image DNA replicationsystem.

Base Complementary Pairing Specificity of Mirror-Image DNA ReplicationSystem

It was further desirable to detect whether the mirror-image polymerasecatalyzed DNA amplification complied with the rule of base complementarypairing. Four kinds of dNTPs were separately added to the reactionsystem, and the extension was detected when the next base of thetemplate was A, T, C, and G (FIG. 8). For the natural system, we usedprimer12 and four different templates, of which the 13^(th) base at the3′ end was A, T, C, and G, respectively. For the mirror-image system, inorder to reduce the cost of purchasing the mirror-image DNA, theextension of 1, 2, 3 nt was performed with primer12, and primer13,primer14, and primer15 were obtained by recovering from PAGE gel, whichwere complementary to template18, and the next base in the template wasT, G, A, and C, respectively. It was observed that only under the fourcorrect pairing conditions of A:T, T:A, C:G, G:C, dNTP can beefficiently added to the 3′ end of the primer sequence without obviousmismatch, consistent in both natural and mirror-image systems.Therefore, the mirror-image DNA replication system also followed thecomplementary pairing rules and has a certain degree of fidelity.

Chiral Specificity of Mirror-Image DNA Replication System

Regarding the chiral specificity of polymerase when adding dNTPs, somestudies have been conducted on natural polymerases. Previous studieshave shown that DNA extensions catalyzed by mammalian polymerase γ, E.coli polymerase I, and HIV-1 reverse transcriptase-catalyzed areinhibited by L-dTTP, whereas for mammalian polymerase α, L-dTTP does notinhibit the DNA polymerization catalyzed by it, and for mammalian β, theinhibition effect is weak (Yamaguchi et al., 1994). L-dTTP inhibitshuman DNA polymerase γ, δ, bovine thymus terminal transferase, but doesnot inhibit human DNA polymerase β, however DNA polymerases β, γ, δcannot use L-dTTP as substrate. The addition of L-nucleoside at the 3′end renders DNA difficult to be degraded by 3′-5′ exonuclease (Focher etal., 1995). Therefore, some polymerases are inhibited by mirror-imagedNTPs when adding two substrates, and some may not. Our study hopes todemonstrate whether there is chiral specificity for mirror-image ASFVpol X and whether its polymerase activity is inhibited by natural chiraldNTPs. In our study, we first tried to add a combination of differentchiral polymerases, primer-templates, and dNTPs separately, and thentried to react both the natural and mirror-image systems in the samesolution.

The chirality of ASFV pol X, primer-template and dNTPs was changed underthe same buffer conditions as before. There are two types of chirality,L-form and D-form, for each component and there were totally 8combinations in the system (FIG. 9). The system was incubated at 37° C.for 12 hours and the results were observed on a 20% PAGE gel. It wasfound that the natural system of L-ASFV pol X, D-primer-template, andD-dNTPs could extend from 6 bases to full-length, and the mirror-imagesystem of D-ASFV pol X, L-primer template and L-dNTPs could extend tothe full-length, but no other combination could extend. For example,L-ASFV pol X, D-primer-template, L-dNTPs, and natural polymerase couldnot add mirror-image dNTPs to the 3′ end of the natural primer; L-ASFVpol X, L-primer-template, D-dNTPs, and natural polymerase could not addnative dNTPs to the 3′ end of the mirror-image primer; D-ASFV pol X,L-primer-template, D-dNTPs, and mirror-image polymerase could not addnative dNTPs to the 3′ end of the mirror-image primer; D-ASFV pol X,D-primer-template, L-dNTPs, and mirror-image polymerase could not addmirror-image dNTPs to the 3′ end of the native primer. Therefore, in themirror-image and natural systems, the primer-template and dNTPs withwrong chirality could not be used as substrate to participate in theextension reaction, and the natural and mirror-image DNA replicationsystems had good chiral specificity.

Furthermore, it was desirable to explore whether natural andmirror-image systems were interfering with each other in the samesolution system. The previously described literature indicated that forsome polymerases, mirror-image dNTPs prevented the DNA extensionreaction from proceeding. Our experiments used the same buffer system asbefore, with the addition of identical concentrations of ASFV pol X anddNTPs of both kinds of charilty. In order to separately observe theextension of the primers, we designed primers and template sequences ofdifferent lengths, and used different fluorescent modifications. Thenatural reaction used Cy5-labeled primer20 and template26, and themirror-image reaction used FAM-labeled primer12 and template18. Bothchiral primers and templates of the same concentration were added to thesame solution system, and the reaction product was separated anddetected on a 20% PAGE gel after reacting at 37° C. for 4 hours (FIG.10). The red band was the natural reaction between 21 nt and 26 nt,while the green band was the mirror-image reaction between 12 nt and 18nt. The sample on the left was the control group with only the naturalsystem, the middle was the control group with only the mirror-imagesystem, and the right was the experimental group with the sameconcentration of the natural system and mirror-image system in the samesolution system. It can be seen that in the experimental group, bothsystems could extend to the full-length of 18 nt and 26 nt respectively,and the reaction rate of the mixed system was similar to that of thesingle reaction, indicating that the natural system and mirror-imagesystem did not have serious mutual interference in the same solution.

Enzymatic Synthesis and Functional Detection of Mirror-Image DNAzyme

It had been verified that a mirror-image DNA replication system couldrely on a template to synthesize DNA and had a certain basecomplementary pairing specificity. It was further desirable to verifythat the mirror-image DNA sequence synthesized by the mirror-imagepolymerase was biologically active.

In the 1990s, in vitro selection was created and applied. Thistechnology can be used to design and screen with a complex library ofrandom DNA or RNA sequences to obtain functional DNA or RNA molecules,mainly aptamers that bind to a particular target, and catalyticallyactive ribozymes and deoxyribozymes (DNAzymes). Taking nucleic acidaptamer as an example, in vitro screening firstly synthesizes asingle-stranded nucleic acid sequence library containing 20-80 randomsequences with a library complexity of 10¹⁴. The primary sequences ofthe different nucleic acids in the library are different and they formdifferent spatial configurations in solution. The random library isinteracted with the target molecule under suitable conditions to captureDNA or RNA molecules capable of binding to the target, and thesemolecules are subjected to PCR amplification. The affinity of theseamplified molecules for the target is enhanced relative to the originalrandom library, then they are used as a secondary library, followed by asecond round of screening of the target molecule. Thus, a high affinitynucleic acid aptamer sequence can be screened by repeated amplification.

D-ASFV pol X was selected to synthesize an L-DNAzyme sequence and itsdeoxyribozyme activity was verified. R. Breaker and colleagues found andvalidated the activity of multiple DNAzyme sequences in the study ofyear 2013 (Gu et al., 2013). We selected a 44 nt Zn²⁺-dependent DNAzymeas a synthetic target. The designed full-length DNAzyme comprised a 12nt primer sequence at the 5′ end and a complete 44 nt DNAzyme sequence(FIG. 11a ):

(SEQ ID NO: 12) 5′ ACTACGAACGCGGGTGCTACAGCCATAGTTGAGCAATTAGTTGAAGTGGCTGTACA

Its reverse complement sequence was used as a template (10 nt was addedto the 3′ end of the template for easy recovery of the single doublestrand), and the full-length DNAzyme sequence was obtained after 36hours of extension with primer 12 (FIG. 11b ). It was found from thealignment with the marker that the size of the DNAzyme synthesized bythe polymerase was correct. The natural polymerase catalyzed thesynthesis of full-length sequence of DNAzyme at a higher rate than themirror-image system, which may be due to impurities in the mirror-imageprimer, template, and dNTPs. This experiment further demonstrated thatthe extended length of the mirror-image DNA replication system was above44 nt and had no apparent sequence selectivity. The full-length DNAzymegel was further stained and excised, carefully separated from the 66 nttemplate, spread overnight in buffer, and the DNAzyme sequence wasprecipitated using a kit. After dissolving the DNAzyme, the 56 ntDNAzyme sequence was folded into the correct structure by heating at 90°C. and then cooled to room temperature. Then, buffer 2 was added to thereaction system to initiate the reaction. The control buffer 2 contained20 mM Mg²⁺, and the experimental group contained 2 mM Zn²⁺, which werereacted at 37° C. for 36 hours. DNAzymes synthesized by both natural andmirror-image enzymes were able to achieve high efficiency self-splicingin the presence of 2 mM Zn²⁺ ions, and were not self-splicing in controlexperiment lacking Zn²⁺ or adding Mg²⁺ (FIG. 11c ). Therefore, it wasdemonstrated that the DNAzyme sequence synthesized by mirror-image ASFVpol X had self-splicing activity.

DNA Template Dependent Mirror-Image Transcription

It has been verified that the mirror-image DNA polymerase could performprimer extension according to the DNA template, and it was desirable toinvestigate whether the mirror-image DNA can be transcribed into amirror-image RNA. The known X-family DNA polymerase has poor selectivityfor dNTPs and rNTPs, pol μ can add dNTPs or rNTPs to the DNA strandaccording to the template. Quantitatively, its selective specificity forrNTP is 1000 times lower than that of pol β (Sa and Ramsden, 2003). Ithas not been verified whether ASFV pol X could utilize rNTP. First, rNTPwas added to the system of primer12 and template18 using the naturalchiral ASFV pol X. It was found that although the efficiency of theextension was much reduced, a full-length extension product was stillobtained after 36 hours (FIG. 12a ).

RNase A specifically catalyzes the cleavage of the next nucleotide 5′phosphodiester bond of the ribose of RNA at the C and U residues, whichrecognizes the 3′,5′ phosphodiester bond but does not cleave the 2′,5′bond, forming a 3′C or 3′U oligonucleotide having a 2′,3′-cyclicphosphate derivative. The extension product after 36 hours of reactionwas heated to 75° C. for 10 minutes, RNase inhibitor and ASFV pol X wereinactivated, and then different concentrations of RNase A were added fordigestion (FIG. 12b ). It can be seen that the heating reaction in thefirst step did not degrade the RNA, but the addition of RNase A causedthe full-length 18 nt product to degrade to 13 nt, and the higher theconcentration of RNase A was, the more obvious the degradation was. Thisexperiment demonstrated that the digestion of RNA synthesis product byRNase A verified that it was rNTP that was added, and in a manner of3′,5′ linkage.

In mirror-image ASFV pol X and DNA system, L-rNTPs could also be addedto the primer strand, albeit at a slower rate than the native system(FIG. 12a ). It was also believed that this might be caused byinsufficient purity of the mirror-image template, primer, and rNTPs(impurity peaks could be observed in HPLC, and the amounts of templateand primer found in CD were less than the nominal values).

Base Complementary Pairing Specificity of Mirror-Image RNA TranscriptionSystem

Finally, it was desirable to verify whether the RNA transcription systemfollowed the base complementary pairing rules according to the template.Four kinds of rNTPs were separately added to the reaction system, andthe 5′-end FAM-labeled primer12 was used as a primer. It was observedwhether it could be extended to 13 nt when the next base of the templatewas A, T, C, and G, respectively. For the natural system, primer12 andfour different templates were used, with the 13^(th) base at the 3′ endbeing A, T, C, and G, respectively. For the mirror-image system, theextension reaction was carried out by adding A, AC and ACT to primer12,and the PAGE gel was recovered to obtain primer13, primer14 andprimer15. When they were complementary to template18, the next base onthe template was T, G, A, and C, respectively. It was observed that RNTPcould be efficiently added to the 3′ end of the primer sequence underthe four correct pairing conditions of A:U, U:A, C:G, and G:C. Someobvious mismatches occurred in the mirror-image system, such as T:rG,A:rC, and the like, but the efficiency of false nucleoside addition wassignificantly lower than the correct pairing (FIG. 13). Therefore, themirror-image RNA transcription system followed the principle ofcomplementary pairing and had a certain degree of fidelity.

BRIEF SUMMARY

The designed and constructed mirror-image chiral genetic informationreplication and transcription system comprised core components D-ASFVpol X, L-primer, L-template, L-dNTPs/rNTPs, which could be used toamplify DNA and transcribe into RNA according to the template,indicating that the mirror-image chiral polymerase interacts with DNA.Using ASFV pol X polymerase, theoretical multi-cycle amplification ofDNA was achieved, and 2.3-fold DNA amplification was achieved from thesecond cycle to the third cycle. In addition, it was verified that theaddition of dNTP or rNTP in the natural and mirror-image system toextend the primer strand follows the base complementary pairing rulesand has good fidelity. The polymerase, primer template and dNTPs hadchiral specificity in the reaction system. Only when three of them wereall natural or all mirror-image molecules, the primer strand could beextended, and other combinations could not achieve amplification. Twochiral DNA replication systems of the same concentration were added tothe same solution system, and the two systems were allowed to carry outextension reactions separately without serious mutual hindrance. The 44nt Zn²⁺-dependent DNAzyme sequence was extended with mirror-image ASFVpol X, and the purified single-stranded L-DNAzyme had self-splicingbiological activity in a buffer environment containing 2 mM Zn²⁺.

SUMMARY AND PROSPECTS Summary

Originating from the exploration of the existence and biologicalactivity of mirror-image biomolecules, our work constructed amirror-image genetic information replication and transcription systembased on D-ASFV pol X. The main conclusions were as follows:

I. A system for mirror-image DNA replication was designed, comprising amirror-image polymerase, a mirror-image DNA, four mirror-image dNTPs,and appropriate buffer conditions (50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂,1 mM DTT, and 50 mM KCl). The full-length sequence of D-ASFV pol X wasobtained by a total chemical synthesis method, and the foldingrenaturation was successfully carried out by a guanidine hydrochloridedenaturation-dialysis method. It was analyzed by HPLC, SDS-PAGE, ESI-MS,CD and the like.

II. In the mirror-image DNA replication system, 0.7 μg of D-ASFV pol Xpolymerase, 2.5 μM L-template, 2.5 μM L-primer and 0.2 mM L-dNTPs (theconcentration for each kind) were added under the above bufferconditions. It was confirmed that replication extension of themirror-image DNA in multiple template primer combinations(primer12-template18, primer15-template21, primer12-DNAzymeTemplate) waspossible. And in the DNA replication process, the newly synthesized DNAwas complementary to the template strand, and the mirror-imagereplication process followed the base complementary pairing rules andhad good fidelity.

III. Multi-cycle amplification of DNA template could be achieved byadding appropriate template and double-stranded primer to the reactionsystem, using a PCR cycle of denaturation at 95° C.—annealing at roomtemperature—extension at 37° C., with additional D-ASFV pol X after eachannealing.

IV. In the experiment, the chiral combination of ASFV pol X,primer-template and dNTPs was changed. There were 8 combinations intotal, and the extension reaction was tried in a suitable reactionsystem. Only the all natural combination of L-ASFV pol X,D-primer-template, and D-dNTPs, as well as the all mirror-imagecombination of D-ASFV pol X, L-primer-template, and L-dNTPs were able toreact, and no extension was detected during the reaction time for theremaining six combinations. It was indicated that the natural andmirror-image DNA replication system based on ASFV pol X can recognizethe substrate of the wrong chirality and has good chiral specificity inthe extension reaction.

V. In the experiment of mixing two chiralities, ASFV pol X, a primer, atemplate and dNTPs of two chiralities were added to the same reactionsystem. Each of the two systems can correctly identify the protein ornucleic acid molecule of its own system and complete the extension ofthe DNA strand respectively, without serious mutual interference.

VI. A 44 nt Zn²⁺-dependent DNAzyme sequence was amplified using amirror-image DNA replication system and a full-length DNA strand wasobtained after 36 hours of extension. The single-stranded DNAzyme wasrecovered and isolated from PAGE gel. In a buffer environment containing2 mM Zn²⁺, the L-DNAzyme had self-splicing biological activity, whilethe control group could not slice under the condition of 20 mM Mg²⁺. Themirror-image DNA replication system can amplify long-chain, active L-DNAsequences.

VII. The mirror-image transcription system comprised D-ASFV pol X, anL-template, an L-primer and L-rNTPs. L-rNTPs can be added to the 3′ endof the template under the conditions of 50 mM Tris-HCl, pH 7.5, 20 mMMgCl₂, 1 mM DTT, and 50 mM KCl to synthesize an RNA sequence. Thereaction rate of D-ASFV pol X template-dependent RNA transcription waslower than that of replicating DNA. The transcription also followed thebase-complementary pairing rules, and some mismatches were produced.

Our work enabled the construction of a mirror-image DNA replication andtranscription system and demonstrated that the mirror-image polymerasecan replicate a mirror-image DNA strand or generate a RNA strandaccording to the template. Although the evolutionary evidence of theearly origins of life has not been found and it is still unknown why themirror-image molecules have been abandoned in the evolution of highercell life, it is experimentally confirmed that the mirror-image lifemolecules can achieve the corresponding biological function, providingthe possibility of the existence of the mirror-image molecules. Therealization of two important aspects in the mirror-image center rulelays the foundation for building a complete mirror-image life in thelaboratory environment in the future.

Applications and Prospects

Our work continued the study of mirror-image chiral biomolecules, notonly confirming that the mirror-image molecule has the activity andchiral specificity corresponding to the natural molecule as theresearchers expected, but also as the beginning of theoretical andpractical applications, completing the two steps in the mirror-imagecenter rule: DNA replication and RNA transcription. The presentinvention has two main fields of application: one is to use amirror-image DNA replication system for screening nucleic acid drugs invitro, and the other is to attempt to construct a complete mirror-imageprimitive cell.

I. The in vitro mirror-image screening of a nucleic acid drug has apromising future. Both natural and mirror-image nucleic acid sequencescan be folded into complex and diverse structures due to their diversesequences, and some DNA or RNA can bind to specific target molecules andare called an aptamer. Generally, the method for obtaining aptamer is anin vitro screening technique, which uses DNA or RNA containing usually30-80 random sequences, to generally achieve a complexity of 10¹⁴ ormore, with a fixed primer complementary region at both ends tofacilitate PCR amplification. In the screening process, the targetmolecule is usually fixed by different methods, and the random sequencelibrary is placed together with the target molecule for binding, andthen the nucleic acid sequence not bound to the target molecule isseparated by the washing liquid, and then the nucleic acid with strongaffinity is obtained, and the sequence with high affinity intensity isenriched by PCR amplification. After multiple cycles of screening, oneor several nucleic acid sequences that bind strongly to the targetmolecule can be obtained.

If in vitro screening is performed for a disease-related protein orsmall molecule, such as CDK, GPCR, Bcl-2, etc., it is possible to obtaina nucleic acid aptamer molecule with high affinity for it. However, theeffects of clinical application of these molecules are not ideal, andthey will be rapidly degraded in the body. Further, researchers hope touse a more stable mirror-image nucleic acid molecule as a drug in thebody. At present, existing studies have carried out in vitro screeningby synthesizing a mirror-image target molecule and using a naturalrandom sequence library to obtain a natural nucleic acid sequence thatcan be combined with a mirror-image target. Due to the chiralmirror-image relationship, the same sequence can be used to synthesizethe mirror-image genomic aptamer to bind to the natural target (Williamset al., 1997). This screening method can effectively obtain themirror-image nucleic acid aptamer that can bind a natural molecule, butthe problem is that the technology of protein chemical synthesis is verydifficult, and few academic or commercial organizations can synthesizecomplex proteins, and most of the targets in living organisms are beyondthe current technical range of protein synthesis, for example, PD-L1protein has a size of 40 kDa.

The difficulty of mirror-image in vitro screening is that there iscurrently no way to perform mirror-image PCR amplification in each roundof screening. This will be realized by our mirror-image PCR technology.A library of mirror-image random sequences is synthesized by a DNAsynthesizer and used directly for the binding of natural drug target. Byrinsing, eluting, and amplifying the high affinity sequence, themirror-image nucleic acid aptamer sequence can be directly obtainedthrough multiple cycles, and can be optimized and clinically tested aspotential drug molecule. For mirror-image PCR technology, mirror-imagePCR is implemented herein by adding ASFV pol X in each cycle. ASFV pol Xhas a very low reaction rate (mainly used for repair of genomic gap invirus), and it is not a thermostable polymerase and loses activity at50° C. for 1 minute under 3.5 M proline-protected condition. In thefuture, a highly efficient, thermostable polymerase that has beendiscovered, such as Dpo4 of 352 amino acids (Sulfolobus solfataricus P2DNA polymerase IV), may be used. It is reported in the literature thatDpo4 may be used for PCR amplification, and PCR and mirror-image invitro screening may be performed using the mirror-image Dpo4 protein.

II. An important step in building a mirror-image life is to implementanother important step in the mirror-image center rule, the translationprocess, in the lab. The mirror-image ribosome and tRNA are the primarybiological primitives for translation, and current state of the artstill tends to construct using chemical synthesis method. The ribosomesof bacteria contain rRNA, usually with 50-80 ribosomal proteins (Wilsonet al., 2009), most of which are shorter than 240 amino acids and can beachieved by current protein synthesis technology. There is also a rpsAprotein of 557 amino acids that needs to be achieved by future improvedprotein synthesis techniques. After each component is synthesized, theyare renatured and assembled in vitro to become a functional mirror-imageribosome. By chemically synthesizing DNA and PCR amplification of alonger L-DNA template, a long L-mRNA is transcribed, and themirror-image ribosome and tRNA can be used to obtain a protein moleculethat realizes the functions of a mirror-image cell, such as DNA ligase,helicase, pyruvate dehydrogenase and the like. If the protein andnucleic acid components required for a mirror-image cell are synthesizedas much as possible, further technological advances will make itpossible to construct simple mirror-image cells, or to use themirror-image cells to produce mirror-image drugs or mirror-imagebiomaterials.

Main symbol comparison table ASFV pol X African Swine Fever VirusPolymerase X DNAzyme Deoxyribozyme SDS-PAGE Sodium dodecyl sulfatepolyacrylamide gel electropheresis Tris TrishydroxymethylaminomethaneNMDA N-methyl-D-aspartic acid HPLC High performance liquidchromatography ESI-MS Electrospray ionization mass spectrometry EDTAEthylene diamine tetraacetic acid FAM Fluorescein amidite

REFERENCES

-   Breslow, R., and Levine, M. (2006). Amplification of enantiomeric    concentrations under credible prebiotic conditions. Proceedings of    the National Academy of Sciences of the United States of America    103, 12979-12980.-   Corrigan, J. (1969). D-Amino Acids in Animals. Science 164, 142-149.-   Dawson, P. E., and Kent, S. B. H. (2000). Synthesis of Native    Proteins by Chemical Ligation. Annual Review of Biochemistry 69,    923-960.-   Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. (1994).    Synthesis of proteins by native chemical ligation. Science 266,    776-779.-   Engel, M., and Macko, S. (2001). The stereochemistry of amino acids    in the Murchison meteorite. Precambrian Research 106, 35-45.-   Fields, G. (2002). Introduction to Peptide Synthesis, Vol Chapter    11.-   Fitzgerald, M. C., Chernushevich, I., Standing, K. G., Kent, S. B.    H., and Whitman, C. P. (1995). Total Chemical Synthesis and    Catalytic Properties of the Enzyme Enantiomers L- and    D-4-Oxalocrotonate Tautomerase. Journal of the American Chemical    Society 117, 11075-11080.-   Focher, F., Maga, G., Bendiscioli, A., Capobianco, M., Colonna, F.,    Garbesi, A., and Spadari, S. (1995). Stereospecificity of human DNA    polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse    transcriptase, HSV-1 DNA polymerase, calf thymus terminal    transferase and Escherichia coli DNA polymerase I in recognizing D-    and L-thymidine 5′-triphosphate as substrate. Nucleic Acids Research    23, 2840-2847.-   Gu, H., Furukawa, K., Weinberg, Z., Berenson, D. F., and    Breaker, R. R. (2013). Small, Highly Active DNAs That Hydrolyze DNA.    Journal of the American Chemical Society 135, 9121-9129.-   Hamase, K., Inoue, T., Morikawa, A., Konno, R., and Zaitsu, K.    (2001). Determination of Free [formula omitted]-Proline and [formula    omitted]-Leucine in the Brains of Mutant Mice Lacking [formula    omitted]-Amino Acid Oxidase Activity. Analytical Biochemistry 298,    253-258.-   Hashimoto, A., Kumashiro, S., Nishikawa, T., Oka, T., Takahashi, K.,    Mito, T., Takashima, S., Doi, N., Mizutani, Y., Yamazaki, T., et al.    (1993). Embryonic Development and Postnatal Changes in Free    d-Aspartate and d-Serine in the Human Prefrontal Cortex. Journal of    Neurochemistry 61, 348-351.-   Hashimoto, A., Oka, T., and Nishikawa, T. (1995). Anatomical    Distribution and Postnatal Changes in Endogenous Free D-Aspartate    and D-Serine in Rat Brain and Periphery. European Journal of    Neuroscience 7, 1657-1663.-   Joyce, G., Visser, G., Van Boeckel, C., Van Boom, J., Orgel, L., and    Van Westrenen, J. (1984). Chiral selection in poly(C)-directed    synthesis of oligo (G). Nature 310, 602.-   Kent, S. B. (1988). Chemical synthesis of peptides and proteins.    Annual Review of Biochemistry 57, 957-989.-   Klusmann, S., Nolte, A., Bald, R., Erdmann, V., and Furste, J.    (1996). Mirror-image RNA that binds D-adenosine. Nature    Biotechnology 14, 1112.-   Krause, E., Bienert, M., Schmieder, P., and Wenschuh, H. (2000). The    Helix-Destabilizing Propensity Scale of d-Amino Acids: The Influence    of Side Chain Steric Effects.-   Liu, S., Pentelute, B. L., and Kent, P. S. B. H. (2012). Convergent    Chemical Synthesis of [Lysine 24,&thinsp; 38,&thinsp; 83] Human    Erythropoietin &dagger. Angewandte Chemie International Edition 51,    993&ndash; 999.-   Maciejewski, M., Shin, R., Pan, B., Marintchev, A., Denninger, A.,    Mullen, M., Chen, K., Gryk, M., and Mullen, G. (2001). Solution    structure of a viral DNA repair polymerase. Nature Structural &    Molecular Biology 8, 936.-   Milton, R., Milton, S., and Kent, S. (1992). Total chemical    synthesis of a D-enzyme: the enantiomers of HIV-1 protease show    reciprocal chiral substrate specificity [corrected]. Science 256,    1445-1448.-   Mothet, J., Parent, A., Wolosker, H., Brady, R., Linden, D., Ferris,    C., Rogawski, M., and Snyder, S. (2000). d-Serine is an endogenous    ligand for the glycine site of the N-methyl-d-aspartate receptor.    Proceedings of the National Academy of Sciences of the United States    of America 97, 4926.-   Nagata, Y., Homma, H., Lee, J., and Imai, K. (1999). d-Aspartate    stimulation of testosterone synthesis in rat Leydig cells. FEBS    Letters 444, 160-164.-   Nolte, A., Klusmann, S., Bald, R., Erdmann, V., and Furste, J.    (1996). Mirror-design of L-oligonucleotide ligands binding to    L-arginine. Nature Biotechnology 14, 1116.-   Ohide, H., Miyoshi, Y., Maruyama, R., Hamase, K., and Konno, R.    (2011). d-Amino acid metabolism in mammals: Biosynthesis,    degradation and analytical aspects of the metabolic study. Journal    of Chromatography B 879, 3162-3168.-   Oliveros, M., Yanez, R., Salas, M., Salas, J., Vinuela, E., and    Blanco, L. (1997). Characterization of an African Swine Fever Virus    20-kDa DNA Polymerase Involved in DNA Repair. Journal of Biological    Chemistry 272, 30899-30910.-   Qian, W., and Danishefsky, S. J. (2007). Free-radical-based,    specific desulfurization of cysteine: a powerful advance in the    synthesis of polypeptides and glycopolypeptides. Angewandte Chemie    International Edition 46, 9248-9252.-   Sa, N., and Ramsden, D. (2003). Polymerase mu is a DNA-directed    DNA/RNA polymerase. Molecular and Cellular Biology 23, 2309-2315.-   Schell, M., Molliver, M., and Snyder, S. (1995). D-serine, an    endogenous synaptic modulator: localization to astrocytes and    glutamate-stimulated release. Proceedings of the National Academy of    Sciences of the United States of America 92, 3948-3952.-   Sczepanski, J., and Joyce, G. (2014). A cross-chiral RNA polymerase    ribozyme. Nature 515, 440.-   Showalter, A., Byeon, I., Su, M., and Tsai, M. (2001). Solution    structure of a viral DNA polymerase X and evidence for a mutagenic    function. Nature Structural & Molecular Biology 8, 942.-   Showalter, A., and Tsai, M. (2001). A DNA Polymerase with    Specificity for Five Base Pairs.-   Vinogradov, A., Evans, E., and Pentelute, B. (2015). Total synthesis    and biochemical characterization of mirror image barnase. Chemical    Science 6, 2997-3002.-   Weinstock, M., Jacobsen, M., and Kay, M. (2014). Synthesis and    folding of a mirror-image enzyme reveals ambidextrous chaperone    activity. Proceedings of the National Academy of Sciences of the    United States of America 111, 11679-11684.-   Williams, K., Liu, X., Schumacher, T., Lin, H., Ausiello, D., Kim,    P., and Bartel, D. (1997). Bioactive and nuclease-resistant 1-DNA    ligand of vasopressin. Proceedings of the National Academy of    Sciences of the United States of America 94, 11285-11290.-   Wilson, D., Gupta, R., Mikolajka, A., and Nierhaus, K. (2009).    Ribosomal Proteins: Role in Ribosomal Functions, Vol 2009.-   Wolosker, H., Blackshaw, S., and Snyder, S. (1999). Serine racemase:    A glial enzyme synthesizing d-serine to regulate    glutamate-N-methyl-d-aspartate neurotransmission. Proceedings of the    National Academy of Sciences of the United States of America 96,    13409-13414.-   Wyszko, E., Szymanski, M., Zeichhardt, H., Muller, F., Barciszewski,    J., and Erdmann, V. (2013). Spiegelzymes: Sequence Specific    Hydrolysis of L-RNA with Mirror Image Hammerhead Ribozymes and    DNAzymes. PloS one 8, e54741.-   Yamaguchi, T., Iwanami, N., Shudo, K., and Saneyoshi, M. (1994).    Chiral Discrimination of Enantiomeric 2′-Deoxythymidine    5′-Triphosphate by HIV-1 Reverse Transcriptase and Eukaryotic DNA    Polymerases. Biochemical and Biophysical Research Communications    200, 1023-1027.-   Zuker, M. (2003). Mfold web server for nucleic acid folding and    hybridization prediction. Nucleic Acids Research 31, 3406-3415.

Example 2

Total Chemical Synthesis of a Thermostable DNA Polymerase

Polymerase chain reaction (PCR) is an important tool for modernbiological research. To achieve a polymerase chain reaction of themirror-image life system, a thermostable DNA polymerase is designed andchemically synthesized. The enzyme is DNA polymerase IV (Dpo4) fromSulfolobus solfataricus P2 strain consisting of 352 amino acid residues.This chemically synthesized L-DNA polymerase is the synthetic proteinwith the largest molecular weight reported to date. After optimizationof the PCR reaction system, the artificially synthesized L-Dpo4 enzymewas used to amplify a DNA sequence of up to 1.5 Kb. The establishment ofthe chemical synthesis route of L-Dpo4 enzyme has laid a solidfoundation for the later synthesis of D-DNA polymerase suitable formirror-image PCR, which may open up a new path for discovering moremirror-image molecular tools.

In previous study, mirror-image African Swine Fever Virus Polymerase X(D-ASFV pol X) containing 174 D-amino acid residues was chemicallysynthesized, and replication and transcription of mirror-image DNA weresuccessfully achieved by the enzyme [1]. However, due to the poorprocessivity of the ASFV pol X enzyme, only a 44 nt Zn²⁺-dependentself-cleaving L-form deoxyribozyme (DNAzyme) could be obtained by aprimer extension experiment [1]. In addition, since ASFV pol X is notthermostable, it was only applied to a conceptual mirror-image genereplication chain reaction experiment, that is, by supplying freshenzyme in each cycle [1], which made the enzyme unable to efficientlyamplify L-DNA, thereby limiting its use in the mirror-image life system.

In order to achieve efficient amplification of the mirror-image DNA, amore efficient and thermostable mirror-image polymerase may be obtainedby directional engineering of the current L-ASFV pol or by searching forand developing a new synthetic route. The most commonly used Taq DNApolymerase in PCR reactions has 832 amino acid residues, but the largestprotein synthesized by a chemical method to date contains only 312 aminoacid residues, so it is difficult to synthesize Taq DNA polymerase bytotal chemical synthesis. However, the thermostable DNA polymerase IV(Dpo4) from Sulfolobus solfataricus P2 strain not only can PCR-amplify aDNA sequence of up to 1.3 kb [3], but also contains only 352 amino acidresidues, so we began to explore the chemical synthesis route of Dpo4enzyme.

Result:

Design of Total Chemical Synthesis of Mutant Dpo4 Enzyme

Since the peptide chain directly synthesized by solid phase peptidesynthesis (SPPS) generally does not exceed 50 amino acid residues, thenatural chemical ligation (NCL) method [4, 5] was used to ligate shortpolypeptide segments into a long polypeptide segment through naturalpeptide bonds. This method requires a cysteine (Cys) residue at theN-terminus of the ligation site, whereas only one cysteine (Cys31) ispresent in the wild-type (WT) Dpo4 amino acid sequence (FIG. 14a ). Inthe absence of a cysteine ligation site, several alanine (Ala) siteswere searched for in the sequence, replaced by cysteine during chemicalsynthesis, and then cysteine was reconverted to alanine by a non-metalfree radical desulfurization method after the ligation was complete. Inthe original sequence, A42, A155, A229 and A268 can be used as theligation sites for this method. However, even if these four alanineresidues were used as the ligation site, most of the polypeptidesegments were greater than 50 amino acid residues, so in order tointroduce more available ligation sites in the amino acid sequence ofthis enzyme, four point-mutations (S86A, N123A, S207A and S313A) wereintroduced in the original amino acid sequence (FIG. 14b ). In addition,in order to avoid the formation of a dimer of the protein moleculethrough a disulfide bond during the folding process, a C31S mutation wasadditionally introduced. The recombinant polymerase containing thesefive mutations was expressed and purified in E. coli and tested foractivity. The experiment showed that the five-point mutations (C31S,S86A, N123A, S207A and S313A) did not affect PCR activity of the enzyme(FIG. 16b ).

With these five point-mutations, a cyanoguanidine-based natural chemicalligation method (FIG. 15) was used to assemble 9 polypeptide segments(Dpo4-1 to Dpo4-9) in the direction from the C to the N-terminal,thereby achieving the total chemical synthesis of Dpo4-5m polymerase [5,7, 8]. To avoid oxidation of the amino acid side chain during SPPS andprotein ligation, the methionine residues (Met1, Met76, Met89, Met157,Met216 and Met251) originally-presented in the protein were replacedwith norleucine (Nle). Since norleucine is isosteric with methionine,the replacement of methionine by norleucine has little effect on proteinstructure and function [9]. In addition, a histidine tag (His6) wasadded to the N-terminus of the synthetic enzyme to further purify theprotein in subsequent experiments. This resulted in a total length of358 amino acids (352 amino acids of the polymerase plus 6 amino acids ofthe His6 tag; see FIG. 14b ) in the final synthesized polypeptide(Dp4-5m).

Chemical Synthesis of Dpo4-5m

All peptide segments ranging in length from 22 to 52 amino acids weresynthesized by Fmoc-based solid phase peptide synthesis and purified byreverse phase high performance liquid chromatography (RP-HPLC). Duringthe synthesis of peptides by solid phase peptide synthesis, theunmodified peptides Dpo4-1 and Dpo4-3 were found to be highlyhydrophobic and their solubility in aqueous acetonitrile was very low.It has been reported in the literature that the incorporation of anisoacyl dipeptide into a peptide segment can increase the watersolubility of the peptide [10, 11], and a traceless modification can beachieved due to rapid O-to-N acyl shift under natural chemical ligationconditions at pH ˜7 [10, 11]. Thus, an isoacyl dipeptide was insertedbetween Val30-Ser31 (in peptide Dpo4-1) and Ala102-Ser103 (in peptideDpo4-3). The experimental results showed that the incorporation of theisoacyl dipeptide improved not only the solubility of the peptidesegment, but also improved the purity of the polypeptide. In addition,acetamidomethyl (Acm) was also used to protect the N-terminal Cys of thepeptide segments Dpo4-3 and Dpo4-4, thus preventing cyclization of thepeptide segments during ligation and Cys desulfurization [12]. At thesame time, trifluoroacetylthiazolidine-4-carboxylic acid (Tfa-Thz) wasintroduced as a protective group for N-terminal Cys in the peptidesegments Dpo4-5, Dpo4-7 and Dpo4-8. Tfa-Thz is compatible with hydrazideoxidation and can be rapidly converted back to Thz under alkalineconditions. Therefore, the N-terminal Cys unprotected peptide segmentsDpo4-13, Dpo4-14 and Dpo4-17 could be conveniently obtained by a one-potstrategy of natural chemical ligation and Tfa-Thz deprotection (seeAppendix).

By assembling 9 peptide segments in the direction from C-terminal toN-terminal using natural chemical ligation [5, 7, 8], a syntheticpeptide segment containing 358 amino acids was obtained with a finalyield of 16 mg (FIG. 19-32). The molecular weight of the obtainedfull-length peptide was analyzed by analytical reversed-phase highperformance liquid chromatography (HPLC) and electrospray ionizationmass spectrometry (ESI-MS) to be 40797.5 Da, and the theoretical valuewas 40799.9 Da (FIG. 32B, C). The lyophilized peptide segment powder wasdissolved in a denaturing buffer containing 6 M guanidine hydrochloride,and then correct folding of the peptide segment was achieved with theaid of successive dialysis against a series of renaturation bufferscontaining 4 M, 2 M, 1 M and 0 M guanidine hydrochloride. The foldedpolymerase was then heated to 78° C., and the thermally labile peptidesegments and the incorrectly folded full-length peptide segments wereaggregated at this temperature and could be removed byultracentrifugation. The fully-folded synthetic Dpo4-5m polymerase waspresent in the supernatant due to its thermal stability, purified by aNi-NTA column and concentrated by a centrifugal filter. The concentratedDpo4-5m polymerase was analyzed for molecular weight and purity bySDS-PAGE (FIG. 16a ). At the same time, the amino acid sequence wasdetermined by LC-MS/MS mass spectrometry, and two independent massspectrometry sequencing experiments were carried out by treated withtrypsin and pepsin. The results of the two experiments covered 100% ofthe sequences (see Table S2).

PCR Reaction Using Chemically Synthesized Dpo4-5m Polymerase

The PCR activity of Dpo4-5m polymerase prepared by the above method wasfirst detected using a 200 bp template. As shown in FIG. 33, bycomparing the relative amounts of PCR products of different cycle times,it was found that the PCR amplification efficiency was about 1.5, whichwas substantially comparable to that of the wild-type and mutantpolymerases obtained by recombinant expression. Subsequently, the PCRproduct amplified for 35 cycles was cloned into the T vector for Sangersequencing, and the alignment with the original sequence showed thatthere were 7 single base deletions and 19 single base mutations in the22 clones tested (FIG. 34). The result indicated that the cumulativemutation rate after 35 cycles was about 0.9%, which was basicallyconsistent with the replication error rate of wild-type Dpo4 polymerasereported in the literature [3, 14].

Subsequently, Dpo4-5m polymerase was used to amplify DNA sequences ofdifferent lengths. As shown in FIG. 17a , PCR products of differentlengths from 110 bp to 1.0 kb can be amplified in 35 cycles, and the PCRamplification efficiency gradually decreases as the template lengthincreases. In this experiment, the PCR reaction conditions werebasically the same, and the extension time varied with the length of thetemplate. For 110 to 300 bp segments, the extension time was set to 2minutes per cycle, 5 minutes for 400 to 600 bp, and 10 minutes for 700to 1000 bp.

Based on the ability of Dpo4-5m to amplify a 1.0 kb DNA fragment, PCRamplification of a 1.1 kb dpo4 gene fragment was attempted andsuccessfully performed (FIG. 17b ), which was the coding gene for Dpo4polymerase itself. In addition, the 120 bp E. coli 5s rRNA gene rrfB andthe 1.5 kb E. coli 16S rRNA gene rrfC were also successfully amplified.These genes can be used to transcribe the corresponding ribosomal RNA(rRNA) and are therefore important for the assembly of mirror-imageribosomes [15]. The 1.5 kb amplified fragment obtained by this methodexceeded the longest fragment (1.3 kb) amplified by the wild type Dpo4polymerase reported in the prior literature.

Assembly PCR Reaction Using Chemically Synthesized Dpo4-5m Polymerase

Assembled PCR is a method by which long DNA oligonucleotide strands thatcannot be efficiently synthesized by chemical synthesis (usually lessthan 150 nt) can be obtained. This method can be used to obtain longmirror-image gene sequences, considering the lack of available L-DNAtemplate sequences in nature. The 198 bp tC19Z gene was successfullyassembled using Dpo4-5m polymerase, which encodes an in vitro evolvedribozyme with RNA polymerase activity using RNA as a template [17]. TwoPCR primers (tC19Z-F115 and tC19Z-R113) with an overlapping sequence of30 bp were used for PCR amplification reaction. After 20 cycles, boththe experimental group and the negative control group (no enzyme) weredigested with exonuclease I (Exo I), which can only digestsingle-stranded DNA but not double-stranded DNA, so only the assembledfull-length double-stranded DNA remained in the final reaction system.The result of agarose electrophoresis in FIG. 18a showed that thefull-length 198b double-stranded DNA was successfully assembled in thisexperiment, and its product sequence was also verified by sangersequencing.

Meanwhile, an attempt was made to use six short primers between 47 ntand 59 nt in length to perform a three-step assembly PCR foramplification to obtain the 198 bp tC19Z gene sequence. (FIG. 18c ).Such an amplification strategy can be applied in the future in amirror-image PCR system, and a full-length L-form gene sequence can befinally assembled starting from a short oligonucleotide primer strand.In this experiment, the first step was to perform five cycles ofassembly PCR with tC19Z-F1 and tC19Z-R1 primers; the second step was touse the assembled PCR product of the first step as a template to amplify10 cycles with primers tC19Z-F2 and tC19Z-R2; and the last step was touse the product of the second step as a template to amplify 20 cycleswith the primers tC19Z-F3 and tC19Z-R3 to obtain the final full-lengthproduct. Agarose gel electrophoresis analysis showed that theamplification products of lengths of 88 bp, 162 bp and 198 bp wererespectively obtained by each step of the three-step PCR reaction (FIG.18d ), and the final full-length product was also verified by sangersequencing. A one-step PCR reaction was also attempted using six primerssimultaneously, but this method produced multiple by-product bands.

Discuss:

The previously reported ASFV pol X-based mirror-image geneticreplication system lacked processivity and thermostability, making itimpossible to perform efficient PCR amplification experiments to obtainlonger L-DNA sequences [1]. Therefore, in order to realize the PCR ofthe mirror-image system, a mutant Dpo4 polymerase capable of performingPCR reaction was designed and chemically synthesized, which also laid asolid foundation for the synthesis of D-DNA polymerase suitable formirror-image system PCR. The efficient mirror-image PCR system thuscreated can generate many mirror-image molecular tools. For example, achirally inverted PCR system can be used for in vitro screening ofmirror-image nucleic acid aptamers (mirror-image Systematic Evolution ofLigands by Exponential Enrichment, miSELEX) to screen for L-form nucleicacid aptamer that specifically bind to a biological target, and isexpected to be a tool for research and treatment.

A major challenge in the development of mirror-image PCR tools is toobtain long DNA template sequences. The synthetic Dpo4-5m solves thisproblem by using short chemically synthesized oligonucleotides forassembly PCR. However, since the synthetic Dpo4-5m polymerase has a lowamplification efficiency for longer templates, it is still difficult toobtain a mirror-image gene larger than 1 kb such as 16S and 23S rRNA. Inthe future, it may be necessary to develop efficient mirror-image DNA orRNA ligase to solve this problem, for example, through either totalchemical synthesis of a nucleic acid ligase consisting of D-form aminoacids, or utility of a ribozyme having a cross-mirror-image ligaseactivity [20].

Although the final yield of Dpo4-5m polymerase based on the developedchemical synthesis route can reach 16 mg, it is still not a practicalmethod for industrial large-scale production of D-form enzymes. In thefuture, the synthetic route can be further optimized with the use ofother ligation methods [21-23], or a truncated Dpo4 polymerase can bedesigned for total chemical synthesis.

In addition, another strategy for achieving efficient mirror-image PCRis to look for other polymerase systems other than Dpo4 polymerase.Besides its low efficiency of amplification with long DNA sequences,Dpo4 polymerase has another disadvantage of low fidelity with areplication error rate between 4×10⁴ and 8×10⁻³ [3, 14]. mirror-image Amore efficient mirror-image PCR system can be achieved by continuing tosearch for other thermostable DNA polymerases with low molecular weight,or by directed modification of a DNA polymerase with low molecularweight to achieve thermal stability and high amplification efficiency[24,25].

Materials and Methods:

9-Fluorenylmethoxycarbonyl (Fmoc)-Based Solid Phase Peptide Synthesis(Fmoc-SPPS)

All peptide segments were artificially synthesized. Amino acid couplingwas carried out using either 4 eq. Fmoc-amino acid, 4 eq. ethylcyanoglyoxylate-2-oxime (Oxyma) and 4 eq. N,N′-diisopropylcarbodiimide(DIC) in DMF, or 4 eq. Fmoc-amino acid, 3.8 eq.O-(6-chloro-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HCTU) and 8 eq. N,N-diisopropylethylamine (DIEA) inDMF. Air-bath heating was used to accelerate the reaction when needed[26]. Peptide segment Dpo4-9 was synthesized on Fmoc-Thr(tBu)-Wang resin(GL Biochem). Other peptide segments were synthesized on Fmoc-hydrazine2-chlorotrityl chloride resin to prepare peptide hydrazides [27].Val30-Ser31 (in segment Dpo4-1) and Ala102-Ser103 (in segment Dpo4-3)were coupled as isoacyl dipeptides. Tfa-Thz-OH was coupled usingOxyma/DIC activation at room temperature. After the completion ofpeptide chain assembly, the peptide segment was cleaved from the resinusing a cleavage reagent (water/benzylsulfide/triisopropylsilane/1,2-ethanedithiol/trifluoroacetic acid in aratio of 0.5/0.5/0.5/0.25/8.25). After bubbling with nitrogen, thepolypeptide was precipitated by adding ice diethyl ether, and finallycentrifuged to collect a solid precipitate to obtain a crudepolypeptide.

Natural Chemical Ligation (NCL)

The polypeptide segment containing a hydrazide group at C-terminus wasdissolved in an acidified ligation buffer (6 M guanidine hydrochlorideand 0.1 M sodium dihydrogen phosphate, pH 3.0). The mixture was placedin an ice salt bath (−15° C.), 10 to 20 eq. NaNO₂ solution was addedthereto, and then stirred and reacted for 30 minutes. Subsequently, 40eq. 4-mercaptophenylacetic acid (MPAA) and 1 eq. polypeptide segmentwith N-terminal cysteine were then added and the pH of the system wasadjusted to 6.5 at room temperature. After overnight reaction, thesystem concentration was diluted by a factor of two by adding 100 mMtrichloroethyl phosphate (TCEP) buffer, and then reacted at roomtemperature for 1 hour under stirring. The target product was finallyisolated using semi-preparative grade RP-HPLC. The ligation product wasanalyzed by HPLC and ESI-MS and purified by semi-preparative HPLC.

Protein Renaturation and Purification

The lyophilized Dpo4-5m polymerase powder was dissolved in a denaturingbuffer containing 6 M guanidine hydrochloride, followed by dialysisagainst a series of renaturation buffers containing 4 M, 2 M, 1 M and 0M guanidine hydrochloride, respectively, and each step of dialysis wascarried out at 4° C. for 10 hours under gently stirring. Thedenaturation and renaturation buffer also contained 50 mM Tris-HCl (pH7.5), 50 mM NaAc, 1 mM DTT, 0.5 mM EDTA and 16% glycerol. Afterrenaturation, the enzyme was dialyzed against a buffer containing 10 mMpotassium phosphate (pH 7.0), 50 mM sodium chloride, 10 mM magnesiumacetate, 10% glycerol and 0.1% 2-mercaptoethanol. The folded polymerasewas heated to 78° C. to precipitate the thermolabile peptide segment,which was then removed by ultracentrifugation (19,000 rpm) for 40minutes at 4° C. The supernatant was added to Ni-NTA resin (Qiagen) andincubated overnight at 4° C., followed by purification as previouslydescribed, but not further purified using a Mono S column [28]. Afterpurification, the absorbance of the protein at a wavelength of 280 nmwas measured using a spectrophotometer, and the extinction coefficientwas set to 24,058 M⁻¹ cm⁻¹, and the molecular weight was set to 40.8kDa. The purity and yield of the synthesized polymerase (about 100 μg)and recombinant enzyme were finally analyzed using 12% SDS-PAGE.

PCR Reaction Using Chemically Synthesized Dpo4-5m Polymerase

The PCR reactions described herein were carried out in a 20 μl systemcontaining 50 mM HEPES (pH 7.5), 5 mM MgCl₂, 50 mM NaCl, 0.1 mM EDTA, 5mM DTT, 10% glycerol, 3% DMSO, 0.1 mg/ml BSA, 200 μM ultrapure dNTPs,0.5 μM bidirectional primers, 2 nM linear double stranded DNA templateand approximately 300 nM Dpo4-5m polymerase. The PCR program was set todenaturation at 86° C. for 3 minutes; followed by 35 cycles ofdenaturation at 86° C. for 30 seconds, annealing at 58-65° C. for 1minute (annealing temperature depending on primer Tm value), extensionat 65° C. for 2 to 15 minutes (extension time is determined by thelength of the amplicon), and last extension at 65° C. for 5 minutes. Inexperiments in which DNA sequences of different lengths were amplified,the same primers (M13-long-F and M13-long-R, see Table S1) were used forPCR, and the extension time for the templates of 110-300 bp was 2minutes, 5 minutes for the templates of 400-600 bp and 10 minutes forthe templates of 700-1000 bp. After the completion of PCR, the PCRproducts were analyzed by 2% agarose gel electrophoresis and stainedwith GoldView (Solarbio). In an experiment to test the amplificationefficiency and fidelity of Dpo4-5m, a linear double-stranded DNA with alength of 200 bp prepared by Q5 DNA polymerase (New England Biolabs) wasused as a template and the product bands of the first 10 cycles wereanalyzed using Image Lab software (Bio-Rad) to analyze theiramplification efficiency. The PCR product after 35 cycles was purifiedusing the DNA Clean & Concentrator kit (Zymo Research) and then clonedinto the T vector for sanger sequencing to analyze its fidelity.

Assembly PCR Using Synthetic Dpo4-5m

The assembly PCR reaction was carried out using two primers sharing a 30bp overlapping region, with a length of 115 nt and 113 nt (tC19Z-F115and tC19Z-R113), respectively (Supplementary Table S1). No othertemplate was added to the reaction, the target product was the 198 bptC19Z gene, and no enzyme was added to the negative control group. After20 cycles of reaction, the products of the experimental group and thecontrol group were treated with exonuclease I at 37° C. for 10 minutes(10 U per 5 μl of PCR product). In an experiment wherein a three-stepmethod was used to assemble the 198 bp tC19Z gene using six shortprimers, the first step used primers tC19Z-F1 and tC19Z-R1, the secondstep used primers tC19Z-F2 and tC19Z-R2, and the third step used primerstC19Z-F3 and tC19Z-R3. The PCR product of the previous step served as atemplate for the next reaction, accounting for about 5% of the nextreaction system. The numbers of cycles of the first, second, and thirdsteps of the PCR reaction were 5, 10, and 20, respectively. After thecompletion of the PCR reaction, the product was analyzed using 3% highresolution agarose gel electrophoresis and stained with GoldView(Solarbio). The resulting full-length product was purified and recoveredusing the DNA Clean & Concentrator kit (Zymo Research) and then clonedinto the T vector for sanger sequencing.

APPENDIX

Total Chemical Synthesis of Thermostable DNA Polymerase

Material:

Chemicals and Reagents

9-fluorenylmethoxycarbonyl-amino acid GL Biochem (Shanghai) and CSbio(Shanghai) Boc-Cys(Acm)-OH, Boc-Cys(Trt)-OH GL Biochem (Shanghai)6-chlorobenzotriazole-N,N,N′,N′- GL Biochem (Shanghai) tetramethyluronhexafluorophosphate Anhydrous 1-hydroxybenzotriazole GL Biochem(Shanghai) (HOBt) N,N′-diisopropylcarbodiimide (DIC) Adamas Ethylcyanoacetaldehyde-2-oxime Adamas (Oxyma) DL-1,4-dithiothreitol (DTT)Adamas N,N-dimethylformamide (DMF) J&K Scientific Trifluoroacetic acid(TFA) J&K Scientific Triisopropylsilane (TIPS) J&K Scientific1,2-ethanedithiol (EDT) J&K Scientific Benzaldehyde J&K ScientificSodium 2-mercaptoethanesulfonate J&K Scientific (MESNa) Palladiumchloride J&K Scientific 2,2′-azobis[2-(2-imidazolin-2-yl)propane] J&KScientific dihydrochloride (VA-044) N,N-diisopropylethylamine (DIEA)Beijing Ouhe Technology Dichloromethane (DCM) Beijing Chemical IndustryGroup Sodium nitrite Sinopharm Sodium monohydrogen phosphate Sinopharmdodecahydrate Piperidine Sinopharm Ether Sinopharm Acetic acid SinopharmSilver acetate Sinopharm Sodium dihydrogen phosphate dihydrate SinopharmSodium hydroxide Sinopharm Guanidine hydrochloride SinopharmHydrochloric acid Sinopharm Sodium chloride Sinopharm4-mercaptophenylacetic acid (MPAA) Alfa Aesar (Heysham, England)Tris(2-carboxyethyl)phosphine Tianjin Liankuan Fine hydrochloride(TCEP•HCl) Chemical Reduced glutathione (GSH) Acros Organics (Belgium)Acetonitrile (HPLC grade) J. T. Baker (New Jersey, U.S.)Experimental Method:

Reversed-phase high performance liquid chromatography (HPLC) andelectrospray ionization mass spectrometry (ESI-MS)

The product was analyzed and purified by reverse-phase high performanceliquid chromatography using a Shimadzu Prominence HPLC system (solventdelivery unit: LC-20AT), in combination with C4 and C18 columns producedby Welch, and the mobile phase was water and acetonitrile (both contain0.1% TFA). Electrospray ionization mass spectrometry was obtained usinga Shimadzu LCMS-2020 liquid chromatography mass spectrometer.

Liquid Chromatography-Secondary Mass Spectrometry (LC-MS/MS) for PeptideSegment Sequencing

The artificially synthesized Dpo4-5m was purified by 12% SDS-PAGE, andtrypsin or pepsin was added for overnight digestion at 37° C. in thegel, and the extracted peptide segment was analyzed by LC-MS. The samplewas separated by a Thermo-Dionex Ultimate 3000 HPLC system directlycoupled to a Thermo Scientific Q Exactive mass spectrometer and elutedat a flow rate of 0.3 μl/min. The analytical column was a self-madefused silica capillary column (75 μm ID, a length of 150 mm; Upchurch,Oak Harbor) filled with C-18 resin (300 Å, 5 μm, Varian, Lexington,Mass., U.S.). The Q Exactive mass spectrometer operated underdata-dependent acquisition mode by the Xcalibur 2.1.2 software with asingle full-scan mass spectrum in the Orbitrap (300-1800 m/z, 70,000resolution) followed by 20 data-dependent MS/MS scans at 27% normalizedcollision energy.

Annexed table S1|Primer Sequence Primer Sequence M13-long-F5′-GTAAAACGACGGCCAGTGAATTAGAACTCGGT-3′ M13-long-R5′-CAGGAAACAGCTATGACCATGATTACGCCAAGTTT-3′ 5s rRNA(rrfB)-F5′-TGCCTGGCGGCAGTAGCGC-3′ 5s rRNA(rrfB)-R5′-ATGCCTGGCAGTTCCCTACTCTCGC-3′ 16s rRNA(rrsC)-F5′-AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGG-3′ 16s rRNA(rrsC)-R5′-TAAGGAGGTGATCCAACCGCAGGTTCC-3′ dpo4-F5′-ATGATTGTTCTTTTCGTTGATTTTGACTACTTT-3′ dpo4-R5′-AGTATCGAAGAACTTGTCTAATCCTATTGCT-3′ tC19Z-F1155′-GTCATTGAAAAAAAAAGACAAATCTGCCCTCAGAGCTTGAGAACATCTTCGGATGCAGAGGAGGCAGCCTTCGGTGGCGCGATAGCGCCAACGTTCTCAACAGACACCCAATACT-3′ tC19Z-R1135′-GGAGCCGAAGCTCCGGGGATTATGACCTGGGCGTGTCTAACATCGCCTTTTCGTCAGGTGTTATCCCCACCCGCCGAAGCGGGAGTATTGGGTGTCTGTTGAGAACGTTGGCG-3′ tC19Z-F15′-AGAGGAGGCAGCCTTCGGTGGCGCGATAGCGCCAACGTTCTCAA CAGACACCCAATACT-3′tC19Z-R1 5′-CAGGTGTTATCCCCACCCGCCGAAGCGGGAGTATTGGGTGTCTGTTGAGAACGTTGGCG-3′ tC19Z-F25′-CAAATCTGCCCTCAGAGCTTGAGAACATCTTCGGATGCAGAGGA GGCAGCCTTCGGTGG-3′tC19Z-R2 5′-ATTATGACCTGGGCGTGTCTAACATCGCCTTTTCGTCAGGTGTTATCCCCACCCGCCGA-3′ tC19Z-F35′-GTCATTGAAAAAAAAAGACAAATCTGCCCTCAGAGCTTGAGAAC ATCTTCG-3′ tC19Z-R35′-GGAGCCGAAGCTCCGGGGATTATGACCTGGGCGTGTCTAACATC GCC-3′

REFERENCES

-   1. Wang, Z., et al., A synthetic molecular system capable of    mirror-image genetic replication and transcription. Nat Chem, 2016.    8(7): p. 698-704.-   2. Weinstock, M. T., M. T. Jacobsen, and M. S. Kay, Synthesis and    folding of a mirror-image enzyme reveals ambidextrous chaperone    activity. Proceedings of the National Academy of Sciences of the    United States of America, 2014. 111(32): p. 11679-11684.-   3. Boudsocq, F., et al., Sulfolobus solfataricus P2 DNA polymerase    IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass    properties akin to eukaryotic polη. Nucleic Acids Research, 2001.    29(22): p. 4607-4616.-   4. Kent, S. B. H., Total chemical synthesis of proteins. Chemical    Society Reviews, 2009. 38(2): p. 338-351.-   5. Zheng, J.-S., et al., Chemical synthesis of proteins using    peptide hydrazides as thioester surrogates. Nat Protocols, 2013.    8(12): p. 2483-2495.-   6. Wan, Q. and S. J. Danishefsky, Free-radical-based, specific    desulfurization of cysteine: a powerful advance in the synthesis of    polypeptides and glycopolypeptides. Angew Chem Int Ed Engl, 2007.    46(48): p. 9248-52.-   7. Fang, G.-M., et al., Protein Chemical Synthesis by Ligation of    Peptide Hydrazides. Angewandte Chemie International Edition, 2011.    50(33): p. 7645-7649.-   8. Huang, Y.-C., G.-M. Fang, and L. Liu, Chemical synthesis of    proteins using hydrazide intermediates. National Science    Review, 2016. 3(1): p. 107-116.-   9. Dery, L., et al., Accessing human selenoproteins through chemical    protein synthesis. Chemical Science, 2017.-   10. Coin, I., The depsipeptide method for solid-phase synthesis of    difficult peptides. J Pept Sci, 2010. 16(5): p. 223-30.-   11. Liu, F., et al., A Synthetic Route to Human Insulin Using    Isoacyl Peptides. Angewandte Chemie, 2014. 126(15): p. 4064-4068.-   12. Raibaut, L., N. Ollivier, and O. Melnyk, Sequential native    peptide ligation strategies for total chemical protein synthesis.    Chem Soc Rev, 2012. 41(21): p. 7001-15.-   13. Huang, Y. C., et al., Synthesis of l- and d-Ubiquitin by One-Pot    Ligation and Metal-Free Desulfurization. Chemistry, 2016. 22(22): p.    7623-8.-   14. Ling, H., et al., Crystal structure of a Y-family DNA polymerase    in action: a mechanism for error-prone and lesion-bypass    replication. Cell, 2001. 107(1): p. 91-102.-   15. Jewett, M. C., et al., In vitro integration of ribosomal RNA    synthesis, ribosome assembly, and translation. Mol Syst Biol, 2013.    9: p. 678.-   16. Stemmer, W. P., et al., Single-step assembly of a gene and    entire plasmid from large numbers of oligodeoxyribonucleotides.    Gene, 1995. 164(1): p. 49-53.-   17. Wochner, A., et al., Ribozyme-catalyzed transcription of an    active ribozyme. Science, 2011. 332(6026): p. 209-12.-   18. Williams, K. P., et al., Bioactive and nuclease-resistant 1-DNA    ligand of vasopressin. Proceedings of the National Academy of    Sciences, 1997. 94(21): p. 11285-11290.-   19. Yatime, L., et al., Structural basis for the targeting of    complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer. Nat    Commun, 2015. 6: p. 6481.-   20. Sczepanski, J. T. and G. F. Joyce, A cross-chiral RNA polymerase    ribozyme. Nature, 2014. 515(7527): p. 440-442.-   21. Fiala, K. A. and Z. Suo, Pre-steady-state kinetic studies of the    fidelity of Sulfolobus solfataricus P2 DNA polymerase IV.    Biochemistry, 2004. 43(7): p. 2106-15.-   22. Bode, J. W., Emerging methods in amide- and peptide-bond    formation. Curr Opin Drug Discov Devel, 2006. 9(6): p. 765-75.-   23. Englebretsen, D. R., B. Garnham, and P. F. Alewood, A cassette    ligation strategy with thioether replacement of three Gly-Gly    peptide bonds: total chemical synthesis of the 101 residue protein    early pregnancy factor [psi(CH(2)S)28-29,56-57,76-77]. J Org    Chem, 2002. 67(17): p. 5883-90.-   24. Zeng, W., et al., Assembly of synthetic peptide vaccines by    chemoselective ligation of epitopes: influence of different chemical    linkages and epitope orientations on biological activity.    Vaccine, 2001. 19(28-29): p. 3843-52.-   25. Pinheiro, V. B., et al., Synthetic genetic polymers capable of    heredity and evolution. Science, 2012. 336(6079): p. 341-4.-   26. Larsen, A. C., et al., A general strategy for expanding    polymerase function by droplet microfluidics. Nat Commun, 2016.    7: p. 11235.-   27. Huang, Y.-C., et al., Accelerated Fmoc solid-phase synthesis of    peptides with aggregation-disrupting backbones. Organic &    Biomolecular Chemistry, 2015. 13(5): p. 1500-1506.-   28. Huang, Y.-C., et al., Facile synthesis of C-terminal peptide    hydrazide and thioester of NY-ESO-1 (A39 A68) from an Fmoc-hydrazine    2-chlorotrityl chloride resin. Tetrahedron, 2014. 70: p. 2951-2955.

The invention claimed is:
 1. A method for replicating a minor-imagenucleic acid, comprising: carrying out a reaction in the presence of amirror-image nucleic acid polymerase, a mirror- image nucleic acidtemplate, a mirror-image nucleic acid primer, and mirror-imagedNTPs/rNTPs to obtain the mirror-image nucleic acid, wherein saidmirror-image nucleic acid polymerase is in a D-form and selected fromthe group consisting of ASFV pol X of SEQ ID NO: 17 and a Dpo4 proteinof Sulfolobus solfataricus which comprises mutations C31S, S86A, N123A,S207A and S313A and methionines Met1, Met76, Met89, Met157, Met216 andMet251 replaced with norleucine.
 2. The method according to claim 1,wherein the mirror-image nucleic acid, the mirror-image nucleic acidtemplate, the mirror-image nucleic acid primer, and the mirror-imagedNTPs/rNTPs are in L-form.
 3. The method according claim 1, wherein themirror-image nucleic acid is L-DNA.
 4. The method according to claim 1,wherein the reaction is a polymerase chain reaction.
 5. The methodaccording to claim 1, wherein the reaction is carried out in a buffer of50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 50 mM NaCl, 0.1 mM EDTA, 5 mM DTT,10% glycerol, 3% DMSO and 0.1 mg/ml BSA.
 6. A method for screening amirror-image nucleic acid molecule, comprising: contacting a library ofrandom mirror-image nucleic acid sequences with a target molecule undera condition that allows binding of the two; obtaining a mirror-imagenucleic acid molecule that binds to the target molecule; and amplifyingthe mirror-image nucleic acid molecule that binds to the target moleculeby mirror-image PCR using a mirror-image nucleic acid polymerase inD-form selected from the group consisting of ASFV pol X as set forth inSEQ ID NO: 17 and a Dpo4 protein of Sulfolobus solfataricus whichcomprises mutations C31S, S86A, N123A, S207A and S313A and allmethionines Met1, Met76, Met89, Met157, Met216 and Met251 replaced withnorleucine.
 7. The method according to claim 6, wherein after thelibrary of random mirror-image nucleic acid sequences is contacted withthe target molecule, a mirror-image nucleic acid molecule that does notbind to the target molecule is removed by washing to obtain themirror-image nucleic acid molecule that binds to the target molecule. 8.The method according to claim 6, wherein the mirror-image nucleic acidmolecule is L-DNA.
 9. The method according to claim 6, wherein themirror-image PCR is performed in a buffer of 50 mM Tris-HCl, pH 7.5, 5mM MgCl₂, 50 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% glycerol, 3% DMSO and0.1 mg/ml BSA.
 10. The method according to claim 1, wherein the Dpo4protein of Sulfolobus solfataricus comprises a His6 tag.